Biocompatible scaffolds with tissue fragments

ABSTRACT

A biocompatible tissue repair implant or scaffold device is provided for use in repairing a variety of tissue injuries, particularly injuries to cartilage, ligaments, tendons, and nerves. The repair procedures may be conducted with implants that contain a biological component that assists in healing or tissue repair. The biocompatible tissue repair implants include a biocompatible scaffold and particles of living tissue, such that the tissue and the scaffold become associated. The particles of living tissue contain one or more viable cells that can migrate from the tissue and populate the scaffold.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/420,093 filed on Oct. 18, 2002 and entitled“Biocompatible Scaffold With Tissue Fragments,” and to U.S. ProvisionalPatent Application No. 60/419,539 filed on Oct. 18, 2002 and entitled“Biocompatible Scaffold for Ligament or Tendon Repair.”

FIELD OF THE INVENTION

[0002] The present invention relates to biocompatible tissue implantdevices for use in the repair of tissue injuries, as well as methods formaking and using such biocompatible tissue implant devices.

BACKGROUND OF THE INVENTION

[0003] Injuries to soft tissue, such as cartilage, skin, muscle, bone,tendon and ligament, where the tissue has been injured or traumatizedfrequently require surgical intervention to repair the damage andfacilitate healing. Such surgical repairs can include suturing orotherwise repairing the damaged tissue with known medical devices,augmenting the damaged tissue with other tissue, using an implant, agraft or any combination of these techniques.

[0004] One common tissue injury involves damage to cartilage, which is anon-vascular, resilient, flexible connective tissue. Cartilage typicallyacts as a “shock-absorber” at articulating joints, but some types ofcartilage provide support to tubular structures, such as for example,the larynx, air passages, and the ears. In general, cartilage tissue iscomprised of cartilage cells, known as chondrocytes, located in anextracellular matrix, which contains collagen, a structural scaffold,and aggrecan, a space-filling proteoglycan. Several types of cartilagecan be found in the body, including hyaline cartilage, fibrocartilageand elastic cartilage. Hyaline cartilage can appear in the body asdistinct pieces, or alternatively, this type of cartilage can be foundfused to the articular ends of bones. Hyaline cartilage is generallyfound in the body as articular cartilage, costal cartilage, andtemporary cartilage (i.e., cartilage that is ultimately converted tobone through the process of ossification). Fibrocartilage is atransitional tissue that is typically located between tendon and bone,bone and bone, and/or hyaline cartilage and hyaline cartilage. Elasticcartilage, which contains elastic fibers distributed throughout theextracellular matrix, is typically found in the epliglottis, the earsand the nose.

[0005] One common example of hyaline cartilage injury is a traumaticfocal articular cartilage defect to the knee. A strong impact to thejoint can result in the complete or partial removal of a cartilagefragment of various size and shape. Damaged articular cartilage canseverely restrict joint function, cause debilitating pain and may resultin long term chronic diseases such as osteoarthritis, which graduallydestroys the cartilage and underlying bone of the joint. Injuries to thearticular cartilage tissue will not heal spontaneously and requiresurgical intervention if symptomatic. The current modality of treatmentconsists of lavage, removal of partially or completely unattached tissuefragments. In addition, the surgeon will often use a variety of methodssuch as abrasion, drilling or microfractures, to induce bleeding intothe cartilage defect and formation of a clot. It is believed that thecells coming from the marrow will form a scar-like tissue calledfibrocartilage that can provide temporary relief to some symptoms.Unfortunately, the fibrocartilage tissue does not have the samemechanical properties as hyaline cartilage and degrades faster over timeas a consequence of wear. Patients typically have to undergo repeatedsurgical procedures which can lead to the complete deterioration of thecartilage surface. More recently, experimental approaches involving theimplantation of autologous chondrocytes have been used with increasingfrequency. The process involves the harvest of a small biopsy ofarticular cartilage in a first surgical procedure, which is thentransported to a laboratory specialized in cell culture foramplification. The tissue biopsy is treated with enzymes that willrelease the chondrocyte cells from the matrix, and the isolated cellswill be grown for a period of 3 to 4 weeks using standard tissue culturetechniques. Once the cell population has reached a target number, thecells are sent back to the surgeon for implantation during a secondsurgical procedure. This manual labor-intense process is extremelycostly and time consuming. Although, the clinical data suggest long termbenefit for the patient, the prohibitive cost of the procedure combinedwith the traumatic impact of two surgical procedures to the knee, hashampered adoption of this technique.

[0006] One common example of cartilage injury is damage to the menisciof a knee joint. There are two menisci of the knee joint, a medial and alateral meniscus. Each meniscus is a biconcave, fibrocartilage tissuethat is interposed between the femur and tibia of the leg. In additionto the menisci of the knee joint, meniscal cartilage can also be foundin the acromioclavicular joint, i.e., the joint between the clavicle andthe acromion of the scapula, in the sternoclavicular joint, i.e., thejoint between the clavicle and the sternum, and in the temporomandibularjoint, i.e., the joint of the lower jaw. The primary functions ofmeniscal cartilage are to bear loads, to absorb shock and to stabilize ajoint. If not treated properly, an injury to the meniscus, such as a“bucket-handle tear” in the knee joint, may lead to the development ofosteoarthritis. Current conventional treatment modalities for damagedmeniscal cartilage include the removal and/or surgical repair of thedamaged cartilage.

[0007] Another common form of tissue injury involves damage to theligaments and/or tendons. Ligaments and tendons are cords or bands offibrous tissue that contains soft collagenous tissue. Ligaments connectbone to bone, while tendons connect muscle to bone. Tendons are fibrouscords or bands of variable length that have considerable strength butare virtually devoid of elasticity. Ligaments, in contrast, aregenerally pliant and flexible, to allow the ligament tissue to havefreedom of movement, and simultaneously strong and inextensible, toprevent the ligament tissue from readily yielding under applied force.Ligaments and tendons are comprised of fascicles, which contain thebasic fibril of the ligament or tendon, as well as the cells thatproduce the ligament or tendon, known as fibroblasts. The fascicles ofthe tendon are generally comprised of very densely arranged collagenousfibers, parallel rows of elongated fibroblasts, and a proteoglycanmatrix. The fascicles of ligaments also contain a proteoglycan matrix,fibroblasts and collagen fibrils, but the fibrils found in ligamenttissue are generally less dense and less structured than the fibrilsfound in tendon tissue.

[0008] One example of a common ligament injury is a torn anteriorcruciate ligament (ACL), which is one of four major ligaments of theknee. The primary function of the ACL is to constrain anteriortranslation, rotary laxity and hyperextension. The lack of an ACL causesinstability of the knee joint and leads to degenerative changes in theknee such as osteoarthritis. The most common repair technique is toremove and discard the ruptured ACL and reconstruct a new ACL usingautologous bone-patellar, tendon-bone or hamstring tendons. Althoughthis technique has shown long-term clinical efficacy, there is morbidityassociated with the harvest site of the tissue graft. Syntheticprosthetic devices have been clinically evaluated in the past withlittle long-term success. The advantages of a synthetic implant are thatthe patient does not suffer from the donor site morbidity that isassociated with autograft procedures, and that patients having asynthetic implant are able to undergo faster rehabilitation of the knee.These synthetic devices were composed of non-resorbable materials andwere designed to be permanent prosthetic implants. A number of problemswere found during the clinical trials of these implants, such as forexample, synovitis, bone tunnel enlargement, wear debris, and elongationand rupture of the devices. For this reason, autograft reconstruction isstill the widely accepted solution for repairing a ruptured ACL.

[0009] A common tendon injury is a damaged or torn rotator cuff, whichis the portion of the shoulder joint that facilitates circular motion ofthe humerus bone relative to the scapula. The most common injuryassociated with the rotator cuff is a strain or tear to thesupraspinatus tendon. This tear can occur at the insertion site of thesupraspinatus tendon, where the tendon attaches to the humerus, therebypartially or fully releasing the tendon (depending upon the severity ofthe injury) from the bone. Additionally, the strain or tear can occurwithin the tendon itself. Treatment for a strained tendon usuallyinvolves rest and reduced use of the tendon. However, depending upon theseverity of the injury, a torn tendon may require surgical intervention,such as for example, in the case of a full tear of the supraspinatustendon from the humerus. In the case of severe tendon damage, surgicalintervention can involve the repair and/or reattachment of torn tissue,which typically requires a healing and recovery period.

[0010] There is a continuing need in this art for novel surgicaltechniques for the surgical treatment of damaged tissue (e.g.,cartilage, meniscal cartilage, ligaments, tendons and skin) that caneffect a more reliable tissue repair and can facilitate the healing ofinjured tissue. Various surgical implants are known and have been usedin surgical procedures to help achieve these benefits. For example, itis known to use various devices and techniques for creating implantshaving isolated cells loaded onto a delivery vehicle. Such cell-seededimplants are used in an in vitro method of making and/or repairingcartilage by growing cartilaginous structures that consist ofchondrocytes seeded onto biodegradable, biocompatible fibrous polymericmatrices. Such methods require the initial isolation of chondrocytesfrom cartilaginous tissue prior to the chondrocytes being seeded ontothe polymeric matrices. Other techniques for repairing damaged tissueemploy implants having stem or progenitor cells that are used to producethe desired tissue. For example, it is known to use stem or progenitorcells, such as the cells within fatty tissue, muscle, or bone marrow, toregenerate bone and/or cartilage in a patient. The stem cells areremoved from the patient and placed in an environment favorable tocartilage formation, thereby inducing the fatty tissue cells toproliferate and to create a different type of cell, such as for example,cartilage cells.

[0011] There continues to exist a need in this art for novel devices andmethods for making and/or repairing damaged tissue and for hastening thehealing of the damaged tissue.

SUMMARY OF THE INVENTION

[0012] This invention relates to biocompatible tissue implants for usein treating tissue, and the methods for making and using these devices.For example, the tissue implants can be used for the repair and/orregeneration of diseased or damaged tissue. Further, the tissue implantscan be used for tissue bulking, cosmetic treatments, therapeutictreatments, tissue augmentation, and tissue repair. The implants includea biocompatible scaffold that is associated with a suspension containingat least one minced tissue fragment. The biocompatible tissue implantscan also include an additional biological agent and/or an optionalretaining element placed over the suspension of minced tissue.

[0013] The invention also relates to a method of preparing suchbiocompatible tissue implants. The implants are made by providing atleast one biocompatible scaffold and a sample of minced tissue,processing the tissue sample to create a suspension of viable tissuehaving at least one minced tissue fragment, and depositing the tissuesample upon the biocompatible scaffold. In one embodiment, the method ofproducing these implants can include the further step of incubating thetissue-laden scaffold in a suitable environment for a duration and underconditions that are sufficient to effectively allow cells within thetissue sample to populate the scaffold.

[0014] The invention is also directed to a kit to assist in thepreparation of the tissue implants of the present invention. The kits ofthe present invention include a sterile container which houses at leastone biocompatible scaffold, a harvesting tool for collecting a tissuesample from a subject, and one or more reagents for sustaining theviability of the tissue sample. The kit can also include a processingtool for mincing the tissue into tissue particles, or alternatively, theharvesting tool can be adapted to collect the tissue sample and toprocess the sample into finely divided tissue particles. The kit can,optionally, also include a delivery device for transferring the scaffoldfrom the sterile container to a subject for implantation.

[0015] The invention also relates to methods of treating tissue usingthe biocompatible tissue implants of the present invention. Tissuetreatment according to these methods can be performed by providing abiocompatible scaffold and a sample of minced tissue, depositing thetissue sample upon the biocompatible scaffold, and placing thetissue-laden scaffold in a desired position relative to the tissue to betreated. In one embodiment, tissue repair can be achieved by providing abiocompatible scaffold and a sample of minced tissue, depositing thetissue sample in a desired position relative to the tissue injury, andplacing the biocompatible scaffold over the tissue. In anotherembodiment, the method of producing these implants can include thefurther step of incubating the tissue-laden scaffold in a suitableenvironment for a duration and under conditions that are effective toallow cells within the tissue sample to populate the scaffold. In yetanother embodiment, the methods of treating tissue can also include theadditional step of affixing the scaffold in a desired position relativeto the tissue to be treated, such as, for example, by fastening thetissue-laden scaffold in place.

[0016] The present invention is also directed to methods for measuringthe effect(s) of a substance on living tissue. According to this aspectof the invention, the bioimplantable tissue implants of the presentinvention can be used to create tissue constructs that can be contactedwith a test substance so that the effects of the substance on livingtissue can be observed and measured. Thus, the bioimplantable tissueconstructs of the present invention can be used as a biologicalscreening assay to measure the effects of a test substance on livingtissue by examining the effect on various biological responses, such asfor example, the effect on cell migration, cell proliferation anddifferentiation and maintenance of cell phenotype.

[0017] In embodiments in which the implant is used for tissue repair,the tissue repair implant can be used to treat a variety of injuries,such as for example, injuries occurring within the musculoskeletalsystem, such as rotator cuff injuries, ACL ruptures, or meniscal tears,as well as injuries occurring in other connective tissues, such as skinand cartilage. Furthermore, such implants can be used in otherorthopaedic surgical procedures, such as hand and foot surgery, torepair tissues such as ligaments, nerves, and tendons.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The invention will be more fully understood by reference to thefollowing detailed description when considered in conjunction with theaccompanying drawings, in which:

[0019]FIG. 1A is photomicrograph that demonstrates that cells in acartilage tissue sample migrate extensively into a polymer scaffold;

[0020]FIG. 1B is a photomicrograph that demonstrates that the migratingcells of FIG. 1A retain their phenotype and the migrating cells producecellular matrix that stains positive for sulfated glycosaminoglycanusing the Safranin O stain;

[0021]FIG. 2A is a photomicrograph that demonstrates that cells withinthe minced tissue loaded on the biocompatible scaffolds, followingimplantation into SCID mice, have proliferated and filled the entirescaffold;

[0022]FIG. 2B is a photomicrograph that demonstrates that cells withinthe minced tissue, following implantation into SCID mice, arechondrocyte-like and are surrounded by an abundant matrix that stainspositive for Safranin O;

[0023]FIG. 3A is a photomicrograph that illustrates a scaffold loadedwith minced tissue;

[0024]FIG. 3B is a photomicrograph that illustrates a scaffold loadedwith minced tissue and platelet rich plasma (PRP) and demonstrates thatgrowth factors in the PRP are beneficial in promoting the migration ofchondrocyte cells from the minced tissue and in promoting maintenance ofdifferentiated phenotype of the chondrocyte cells within the scaffolds;

[0025]FIG. 4 is a photomicrograph that demonstrates that autologous celldispersion (derived from skin) is present histologically as keratinocyteislands;

[0026]FIG. 5A is a photomicrograph that demonstrates the extensivemigration of cells into the polymer scaffolds after incubating for 6weeks in culture the biocompatible scaffolds having minced anteriorcruciate tissue fragments that have been treated with collagenase;

[0027]FIG. 5B is a photomicrograph that demonstrates the extensivemigration of cells into the polymer scaffolds after incubating for 6weeks in culture the biocompatible scaffolds having minced anteriorcruciate tissue fragments treated without collagenase;

[0028]FIG. 6A is a graph that demonstrates that cells in a meniscalexplant sample migrate extensively into a polymer scaffold;

[0029]FIG. 6B is a photomicrograph that illustrates the histology ofcross sections of the associated meniscal explant and biocompatiblescaffolds, which demonstrates that cells in the meniscal explant samplemigrate into the polymer scaffold.

[0030] FIGS. 7A-7C are photomicrographs of histological sections ofexplant samples obtained following the procedure of Example 7,demonstrating the distribution and nature of tissue formed within ascaffold and grown from minced cartilage tissue fragments.

[0031] FIGS. 8A-8C are photomicrographs of histological sections ofexplant samples obtained following the procedure of Example 7,demonstrating the distribution and nature of tissue formed within ascaffold and grown from bone cartilage paste.

[0032]FIG. 9 is a graph comparing the numbers of cells obtained fordifferent sizes of minced cartilage tissue fragments.

[0033] FIGS. 10A-10C are photomicrographs of histological sections ofexplant samples obtained following the procedure of Example 8,demonstrating the uniformity of the cartilage-like tissue obtained withminced cartilage tissue fragments of different sizes.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The biocompatible tissue implants of the present invention areused in the treatment of various types of tissue for various purposes.For example, the implants can be used for the repair and/or regenerationof diseased or damaged tissue, or they can be used for tissue bulking,tissue augmentation, cosmetic treatments, therapeutic treatments, andfor tissue sealing. The tissue implants include a biocompatible scaffoldand a suspension of minced tissue having at least one minced tissuefragment, wherein the minced tissue suspension is associated with thescaffold. The minced tissue in the suspension of the present inventionincludes at least one viable cell that can migrate from the tissuefragment and onto the scaffold.

[0035] Although the implants are sometimes referred to herein as “tissuerepair implants” and the methods of using the implants are sometimescharacterized as tissue repair techniques, it is understood that theimplants can be used for a variety of tissue treatments, including butnot limited to tissue repair, tissue bulking, cosmetic treatments,therapeutic treatments, tissue augmentation, and tissue sealing.

[0036] The biocompatible tissue implant of the present inventionincludes a biocompatible scaffold having at least a portion in contactwith the minced tissue suspension. The minced tissue suspension can bedisposed on the outer surface of the scaffold, on an inner region of thescaffold, and any combination thereof, or alternatively, the entirescaffold can be in contact with the minced tissue suspension. Thescaffold can be formed using virtually any material or delivery vehiclethat is biocompatible, bioimplantable, easily sterilized and that hassufficient structural integrity and physical and/or mechanicalproperties to effectively provide for ease of handling in an operatingroom environment and to permit it to accept and retain sutures or otherfasteners without substantially tearing. Alternatively, the scaffoldcould be in the form of an injectable gel that would set in place at thedefect site. Sufficient strength and physical properties are developedin the scaffold through the selection of materials used to form thescaffold, and the manufacturing process. Preferably, the scaffold isalso pliable so as to allow the scaffold to adjust to the dimensions ofthe target site of implantation. In some embodiments, the scaffold canbe a bioresorbable or bioabsorbable material.

[0037] In one embodiment of the present invention, the scaffold can beformed from a biocompatible polymer. A variety of biocompatible polymerscan be used to make the biocompatible tissue implants or scaffolddevices according to the present invention. The biocompatible polymerscan be synthetic polymers, natural polymers or combinations thereof. Asused herein the term “synthetic polymer” refers to polymers that are notfound in nature, even if the polymers are made from naturally occurringbiomaterials. The term “natural polymer” refers to polymers that arenaturally occurring. In embodiments where the scaffold includes at leastone synthetic polymer, suitable biocompatible synthetic polymers caninclude polymers selected from the group consisting of aliphaticpolyesters, poly(amino acids), poly(propylene fumarate),copoly(ether-esters), polyalkylenes oxalates, polyamides, tyrosinederived polycarbonates, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, and blends thereof. Suitablesynthetic polymers for use in the present invention can also includebiosynthetic polymers based on sequences found in collagen, elastin,thrombin, fibronectin, starches, poly(amino acid), gelatin, alginate,pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin,hyaluronic acid, ribonucleic acids, deoxyribonucleic acids,polypeptides, proteins, polysaccharides, polynucleotides andcombinations thereof.

[0038] For the purpose of this invention aliphatic polyesters include,but are not limited to, homopolymers and copolymers of lactide (whichincludes lactic acid, D-,L- and meso lactide); glycolide (includingglycolic acid); ε-caprolactone; p-dioxanone (1,4-dioxan-2-one);trimethylene carbonate (1,3-dioxan-2-one); alkyl derivatives oftrimethylene carbonate; δ-valerolactone; β-butyrolactone;γ-butyrolactone; ε-decalactone; hydroxybutyrate; hydroxyvalerate;1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclotetradecane-7,14-dione); 1,5-dioxepan-2-one;6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine; pivalolactone; α,αdiethylpropiolactone; ethylene carbonate; ethylene oxalate;3-methyl-1,4-dioxane-2,5-dione; 3,3-diethyl-1,4-dioxan-2,5-dione;6,6-dimethyl-dioxepan-2-one; 6,8-dioxabicycloctane-7-one and polymerblends thereof. Aliphatic polyesters used in the present invention canbe homopolymers or copolymers (random, block, segmented, tapered blocks,graft, triblock, etc.) having a linear, branched or star structure.Poly(iminocarbonates), for the purpose of this invention, are understoodto include those polymers as described by Kemnitzer and Kohn, in theHandbook of Biodegradable Polymers, edited by Domb, et. al., HardwoodAcademic Press, pp. 251-272 (1997). Copoly(ether-esters), for thepurpose of this invention, are understood to include thosecopolyester-ethers as described in the Journal of Biomaterials Research,Vol. 22, pages 993-1009, 1988 by Cohn and Younes, and in PolymerPreprints (ACS Division of Polymer Chemistry), Vol. 30(1), page 498,1989 by Cohn (e.g., PEO/PLA). Polyalkylene oxalates, for the purpose ofthis invention, include those described in U.S. Pat. Nos. 4,208,511;4,141,087; 4,130,639; 4,140,678; 4,105,034; and 4,205,399.Polyphosphazenes, co-, ter- and higher order mixed monomer basedpolymers made from L-lactide, D,L-lactide, lactic acid, glycolide,glycolic acid, para-dioxanone, trimethylene carbonate and ε-caprolactonesuch as are described by Allcock in The Encyclopedia of Polymer Science,Vol. 13, pages 31-41, Wiley Intersciences, John Wiley & Sons, 1988 andby Vandorpe, et al in the Handbook of Biodegradable Polymers, edited byDomb, et al., Hardwood Academic Press, pp. 161-182 (1997).Polyanhydrides include those derived from diacids of the formHOOC—C₆H₄—O—(CH₂)_(m)—O—C₆H₄—COOH, where “m” is an integer in the rangeof from 2 to 8, and copolymers thereof with aliphatic alpha-omegadiacids of up to 12 carbons. Polyoxaesters, polyoxaamides andpolyoxaesters containing amines and/or amido groups are described in oneor more of the following U.S. Pat. Nos. 5,464,929; 5,595,751; 5,597,579;5,607,687; 5,618,552; 5,620,698; 5,645,850; 5,648,088; 5,698,213;5,700,583; and 5,859,150. Polyorthoesters such as those described byHeller in Handbook of Biodegradable Polymers, edited by Domb, et al.,Hardwood Academic Press, pp. 99-118 (1997).

[0039] As used herein, the term “glycolide” is understood to includepolyglycolic acid. Further, the term “lactide” is understood to includeL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers.

[0040] Elastomeric copolymers are also particularly useful in thepresent invention. Suitable elastomeric polymers include those with aninherent viscosity in the range of about 1.2 dL/g to 4 dL/g, morepreferably about 1.2 dL/g to 2 dL/g and most preferably about 1.4 dL/gto 2 dL/g as determined at 25° C. in a 0.1 gram per deciliter (g/dL)solution of polymer in hexafluoroisopropanol (HFIP). Further, suitableelastomers exhibit a high percent elongation and a low modulus, whilepossessing good tensile strength and good recovery characteristics. Inthe preferred embodiments of this invention, the elastomer exhibits apercent elongation greater than about 200 percent and preferably greaterthan about 500 percent. In addition to these elongation and modulusproperties, suitable elastomers should also have a tensile strengthgreater than about 500 psi, preferably greater than about 1,000 psi, anda tear strength of greater than about 50 lbs/inch, preferably greaterthan about 80 lbs/inch.

[0041] Exemplary biocompatible elastomers that can be used in thepresent invention include, but are not limited to, elastomericcopolymers of ε-caprolactone and glycolide (including polyglycolic acid)with a mole ratio of ε-caprolactone to glycolide of from about 35:65 toabout 65:35, more preferably from 45:55 to 35:65; elastomeric copolymersof ε-caprolactone and lactide (including L-lactide, D-lactide, blendsthereof, and lactic acid polymers and copolymers) where the mole ratioof ε-caprolactone to lactide is from about 35:65 to about 65:35 and morepreferably from 45:55 to 30:70 or from about 95:5 to about 85:15;elastomeric copolymers of p-dioxanone (1,4-dioxan-2-one) and lactide(including L-lactide, D-lactide, blends thereof, and lactic acidpolymers and copolymers) where the mole ratio of p-dioxanone to lactideis from about 40:60 to about 60:40; elastomeric copolymers ofε-caprolactone and p-dioxanone where the mole ratio of ε-caprolactone top-dioxanone is from about from 30:70 to about 70:30; elastomericcopolymers of p-dioxanone and trimethylene carbonate where the moleratio of p-dioxanone to trimethylene carbonate is from about 30:70 toabout 70:30; elastomeric copolymers of trimethylene carbonate andglycolide (including polyglycolic acid) where the mole ratio oftrimethylene carbonate to glycolide is from about 30:70 to about 70:30;elastomeric copolymers of trimethylene carbonate and lactide (includingL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers) where the mole ratio of trimethylene carbonate to lactide isfrom about 30:70 to about 70:30; and blends thereof. Examples ofsuitable biocompatible elastomers are described in U.S. Pat. Nos.4,045,418; 4,057,537 and 5,468,253.

[0042] In one embodiment, the elastomer is a copolymer of 35:65ε-caprolactone and glycolide, formed in a dioxane solvent and includinga polydioxanone mesh. In another embodiment, the elastomer is acopolymer of 40:60 ε-caprolactone and lactide with a polydioxanone mesh.In yet another embodiment, the elastomer is a 50:50 blend of a 35:65copolymer of ε-caprolactone and glycolide and 40:60 copolymer ofε-caprolactone and lactide. The polydioxanone mesh may be in the form ofa one layer thick two-dimensional mesh or a multi-layer thickthree-dimensional mesh.

[0043] The scaffold of the present invention can, optionally, be formedfrom a bioresorbable or bioabsorbable material that has the ability toresorb in a timely fashion in the body environment. The differences inthe absorption time under in vivo conditions can also be the basis forcombining two different copolymers when forming the scaffolds of thepresent invention. For example, a copolymer of 35:65 ε-caprolactone andglycolide (a relatively fast absorbing polymer) can be blended with40:60 ε-caprolactone and L-lactide copolymer (a relatively slowabsorbing polymer) to form a biocompatible scaffold. Depending upon theprocessing technique used, the two constituents can be either randomlyinter-connected bicontinuous phases, or the constituents could have agradient-like architecture in the form of a laminate type composite witha well integrated interface between the two constituent layers. Themicrostructure of these scaffolds can be optimized to regenerate orrepair the desired anatomical features of the tissue that is beingregrown.

[0044] In one embodiment, it is desirable to use polymer blends to formscaffolds which transition from one composition to another compositionin a gradient-like architecture. Scaffolds having this gradient-likearchitecture are particularly advantageous in tissue engineeringapplications to repair or regenerate the structure of naturallyoccurring tissue such as cartilage (articular, meniscal, septal,tracheal, auricular, costal, etc.), tendon, ligament, nerve, esophagus,skin, bone, and vascular tissue. For example, by blending an elastomerof ε-caprolactone-co-glycolide with ε-caprolactone-co-lactide (e.g.,with a mole ratio of about 5:95) a scaffold may be formed thattransitions from a softer spongy material to a stiffer more rigidmaterial, for example, in a manner similar to the transition fromcartilage to bone. Clearly, one of ordinary skill in the art willappreciate that other polymer blends may be used for similar gradienteffects, or to provide different gradients (e.g., different absorptionprofiles, stress response profiles, or different degrees of elasticity).For example, such design features can establish a concentration gradientfor the suspension of minced tissue associated with the scaffolds of thepresent invention, such that a higher concentration of the tissuefragments is present in one region of the implant (e.g., an interiorportion) than in another region (e.g., outer portions).

[0045] The biocompatible scaffold of the tissue repair implant of thepresent invention can also include a reinforcing material comprised ofany absorbable or non-absorbable textile having, for example, woven,knitted, warped knitted (i.e., lace-like), non-woven, and braidedstructures. In one embodiment, the reinforcing material has a mesh-likestructure. In any of the above structures, mechanical properties of thematerial can be altered by changing the density or texture of thematerial, the type of knit or weave of the material, the thickness ofthe material, or by embedding particles in the material. The mechanicalproperties of the material may also be altered by creating sites withinthe mesh where the fibers are physically bonded with each other orphysically bonded with another agent, such as, for example, an adhesiveor a polymer. The fibers used to make the reinforcing component can bemonofilaments, yarns, threads, braids, or bundles of fibers. Thesefibers can be made of any biocompatible material including bioabsorbablematerials such as polylactic acid (PLA), polyglycolic acid (PGA),polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate(TMC), copolymers or blends thereof. These fibers can also be made fromany biocompatible materials based on natural polymers including silk andcollagen-based materials. These fibers can also be made of anybiocompatible fiber that is nonresorbable, such as, for example,polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene),polycarbonate, polypropylene and poly(vinyl alcohol). In one embodiment,the fibers are formed from 95:5 copolymer of lactide and glycolide.

[0046] In another embodiment, the fibers that form the reinforcingmaterial can be made of a bioabsorbable glass. Bioglass, a silicatecontaining calcium phosphate glass, or calcium phosphate glass withvarying amounts of solid particles added to control resorption time areexamples of materials that could be spun into glass fibers and used forthe reinforcing material. Suitable solid particles that may be addedinclude iron, magnesium, sodium, potassium, and combinations thereof.

[0047] The biocompatible scaffolds as well as the reinforcing materialmay also be formed from a thin, perforation-containing elastomeric sheetwith pores or perforations to allow tissue ingrowth. Such a sheet couldbe made of blends or copolymers of polylactic acid (PLA), polyglycolicacid (PGA), polycaprolactone (PCL), and polydioxanone (PDO).

[0048] In one embodiment, filaments that form the biocompatiblescaffolds or the reinforcing material may be co-extruded to produce afilament with a sheath/core construction. Such filaments are comprisedof a sheath of biodegradable polymer that surrounds one or more corescomprised of another biodegradable polymer. Filaments with afast-absorbing sheath surrounding a slower-absorbing core may bedesirable in instances where extended support is necessary for tissueingrowth.

[0049] One of ordinary skill in the art will appreciate that one or morelayers of the reinforcing material may be used to reinforce the tissueimplant of the invention. In addition, biodegradable textile scaffolds,such as, for example, meshes, of the same structure and chemistry ordifferent structures and chemistries can be overlaid on top of oneanother to fabricate biocompatible tissue implants with superiormechanical strength.

[0050] In embodiments where the scaffold includes at least one naturalpolymer, suitable examples of natural polymers include, but are notlimited to, fibrin-based materials, collagen-based materials, hyaluronicacid-based materials, glycoprotein-based materials, cellulose-basedmaterials, silks and combinations thereof. By way of nonlimitingexample, the biocompatible scaffold can be constructed from acollagen-based small intestine submucosa.

[0051] In another embodiment of the present invention, the biocompatiblescaffold can be formed from a biocompatible ceramic material. Suitablebiocompatible ceramic materials include, for example, hydroxyapatite,α-tricalcium phosphate, β-tricalcium phosphate, bioactive glass, calciumphosphate, calcium sulfate, calcium carbonate, xenogeneic and allogeneicbone material and combinations thereof. Suitable bioactive glassmaterials for use in the present invention include silicates containingcalcium phosphate glass, or calcium phosphate glass with varying amountsof solid particles added to control resorption time. Suitable compoundsthat may be incorporated into the calcium phosphate bioactive glassinclude, but are not limited to, magnesium oxide, sodium oxide,potassium oxide, and combinations thereof.

[0052] In yet another embodiment of the tissue implants of the presentinvention, the scaffold can be formed using tissue grafts, such as maybe obtained from autogeneic tissue, allogeneic tissue and xenogeneictissue. By way of non-limiting example, tissues such as skin, cartilage,ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenterand sinew can be used as tissue grafts to form the biocompatiblescaffold. In some embodiments where an allogeneic tissue is used, tissuefrom a fetus or newborns can be used to avoid the immunogenicityassociated with some adult tissues.

[0053] In another embodiment, the scaffold could be in the form of aninjectable gel that would set in place at the defect site. The gel canbe a biological or synthetic hydrogel, including alginate, cross-linkedalginate, hyaluronic acid, collagen gel, fibrin glue, fibrin clot,poly(N-isopropylacrylamide), agarose, chitin, chitosan, cellulose,polysaccharides, poly(oxyalkylene), a copolymer of poly(ethyleneoxide)-poly(propylene oxide), poly(vinyl alcohol), polyacrylate,platelet rich plasma (PRP) clot, platelet poor plasma (PPP) clot,Matrigel, or blends thereof.

[0054] In still yet another embodiment of the tissue implants, thescaffold can be formed from a polymeric foam component having pores withan open cell pore structure. The pore size can vary, but preferably, thepores are sized to allow tissue ingrowth. More preferably, the pore sizeis in the range of about 50 to 1000 microns, and even more preferably,in the range of about 50 to 500 microns. The polymeric foam componentcan, optionally, contain a reinforcing component, such as for example,the textiles disclosed above. In some embodiments where the polymericfoam component contains a reinforcing component, the foam component canbe integrated with the reinforcing component such that the pores of thefoam component penetrate the mesh of the reinforcing component andinterlock with the reinforcing component.

[0055] The foam component of the tissue implant may be formed as a foamby a variety of techniques well known to those having ordinary skill inthe art. For example, the polymeric starting materials may be foamed bylyophilization, supercritical solvent foaming (i.e., as described in EP464,163), gas injection extrusion, gas injection molding or casting withan extractable material (e.g., salts, sugar or similar suitablematerials).

[0056] In one embodiment, the foam component of the engineered tissuerepair implant devices of the present invention may be made by apolymer-solvent phase separation technique, such as lyophilization.Generally, however, a polymer solution can be separated into two phasesby any one of the four techniques: (a) thermally inducedgelation/crystallization; (b) non-solvent induced separation of solventand polymer phases; (c) chemically induced phase separation, and (d)thermally induced spinodal decomposition. The polymer solution isseparated in a controlled manner into either two distinct phases or twobicontinuous phases. Subsequent removal of the solvent phase usuallyleaves a porous structure with a density less than the bulk polymer andpores in the micrometer ranges. See Microcellular Foams Via PhaseSeparation, J. Vac. Sci. Technol., A. T. Young, Vol. 4(3), May/June1986.

[0057] The steps involved in the preparation of these foams includechoosing the right solvents for the polymers to be lyophilized andpreparing a homogeneous solution. Next, the polymer solution issubjected to a freezing and vacuum drying cycle. The freezing step phaseseparates the polymer solution and vacuum drying step removes thesolvent by sublimation and/or drying, leaving a porous polymer structureor an interconnected open cell porous foam.

[0058] Suitable solvents that may be used in the preparation of the foamcomponent include, but are not limited to, formic acid, ethyl formate,acetic acid, hexafluoroisopropanol (HFIP), cyclic ethers (e.g.,tetrahydrofuran (THF), dimethylene fluoride (DMF), and polydioxanone(PDO)), acetone, acetates of C2 to C5 alcohols (e.g., ethyl acetate andt-butylacetate), glyme (e.g., monoglyme, ethyl glyme, diglyme, ethyldiglyme, triglyme, butyl diglyme and tetraglyme), methylethyl ketone,dipropyleneglycol methyl ether, lactones (e.g., γ-valerolactone,δ-valerolactone, β-butyrolactone, γ-butyrolactone), 1,4-dioxane,1,3-dioxolane, 1,3-dioxolane-2-one (ethylene carbonate),dimethlycarbonate, benzene, toluene, benzyl alcohol, p-xylene,naphthalene, tetrahydrofuran, N-methyl pyrrolidone, dimethylformamide,chloroform, 1,2-dichloromethane, morpholine, dimethylsulfoxide,hexafluoroacetone sesquihydrate (HFAS), anisole and mixtures thereof.Among these solvents, a preferred solvent is 1,4-dioxane. A homogeneoussolution of the polymer in the solvent is prepared using standardtechniques.

[0059] The applicable polymer concentration or amount of solvent thatmay be utilized will vary with each system. Generally, the amount ofpolymer in the solution can vary from about 0.5% to about 90% and,preferably, will vary from about 0.5% to about 30% by weight, dependingon factors such as the solubility of the polymer in a given solvent andthe final properties desired in the foam.

[0060] In one embodiment, solids may be added to the polymer-solventsystem to modify the composition of the resulting foam surfaces. As theadded particles settle out of solution to the bottom surface, regionswill be created that will have the composition of the added solids, notthe foamed polymeric material. Alternatively, the added solids may bemore concentrated in desired regions (i.e., near the top, sides, orbottom) of the resulting tissue implant, thus causing compositionalchanges in all such regions. For example, concentration of solids inselected locations can be accomplished by adding metallic solids to asolution placed in a mold made of a magnetic material (or vice versa).

[0061] A variety of types of solids can be added to the polymer-solventsystem. Preferably, the solids are of a type that will not react withthe polymer or the solvent. Generally, the added solids have an averagediameter of less than about 1.0 mm and preferably will have an averagediameter of about 50 to about 500 microns. Preferably, the solids arepresent in an amount such that they will constitute from about 1 toabout 50 volume percent of the total volume of the particle andpolymer-solvent mixture (wherein the total volume percent equals 100volume percent).

[0062] Exemplary solids include, but are not limited to, particles ofdemineralized bone, calcium phosphate particles, bioglass particles,calcium sulfate, or calcium carbonate particles for bone repair,leachable solids for pore creation and particles of bioabsorbablepolymers not soluble in the solvent system that are effective asreinforcing materials or to create pores as they are absorbed, andnon-bioabsorbable materials.

[0063] Suitable leachable solids include nontoxic leachable materialssuch as salts (e.g., sodium chloride, potassium chloride, calciumchloride, sodium tartrate, sodium citrate, and the like), biocompatiblemono and disaccharides (e.g., glucose, fructose, dextrose, maltose,lactose and sucrose), polysaccharides (e.g., starch, alginate,chitosan), water soluble proteins (e.g., gelatin and agarose). Theleachable materials can be removed by immersing the foam with theleachable material in a solvent in which the particle is soluble for asufficient amount of time to allow leaching of substantially all of theparticles, but which does not dissolve or detrimentally alter the foam.The preferred extraction solvent is water, most preferablydistilled-deionized water. Such a process is described in U.S. Pat. No.5,514,378. Preferably the foam will be dried after the leaching processis complete at low temperature and/or vacuum to minimize hydrolysis ofthe foam unless accelerated absorption of the foam is desired.

[0064] Suitable non-bioabsorbable materials include biocompatible metalssuch as stainless steel, cobalt chrome, titanium and titanium alloys,and bioinert ceramic particles (e.g., alumina, zirconia, and calciumsulfate particles). Further, the non-bioabsorbable materials may includepolymers such as polyethylene, polyvinylacetate, polymethylmethacrylate,polypropylene, poly(ethylene terephthalate), silicone, polyethyleneoxide, polyethylene glycol, polyurethanes, polyvinyl alcohol, naturalpolymers (e.g., cellulose particles, chitin, and keratin), andfluorinated polymers and copolymers (e.g., polyvinylidene fluoride,polytetrafluoroethylene, and hexafluoropropylene).

[0065] It is also possible to add solids (e.g., barium sulfate) thatwill render the tissue implants radio opaque. The solids that may beadded also include those that will promote tissue regeneration orregrowth, as well as those that act as buffers, reinforcing materials orporosity modifiers.

[0066] As noted above, porous, reinforced tissue repair implant devicesof the present invention are made by injecting, pouring, or otherwiseplacing, the appropriate polymer solution into a mold set-up comprisedof a mold and the reinforcing elements of the present invention. Themold set-up is cooled in an appropriate bath or on a refrigerated shelfand then lyophilized, thereby providing a reinforced scaffold. Abiological component can be added either before or after thelyophilization step. In the course of forming the foam component, it isbelieved to be important to control the rate of freezing of thepolymer-solvent system. The type of pore morphology that is developedduring the freezing step is a function of factors such as the solutionthermodynamics, freezing rate, temperature to which it is cooled,concentration of the solution, and whether homogeneous or heterogenousnucleation occurs. One of ordinary skill in the art can readily optimizethe parameters without undue experimentation.

[0067] The required general processing steps include the selection ofthe appropriate materials from which the polymeric foam and thereinforcing components are made. If a mesh reinforcing material is used,the proper mesh density must be selected. Further, the reinforcingmaterial must be properly aligned in the mold, the polymer solution mustbe added at an appropriate rate and, preferably, into a mold that istilted at an appropriate angle to avoid the formation of air bubbles,and the polymer solution must be lyophilized.

[0068] In embodiments that utilize a mesh reinforcing material, thereinforcing mesh has to be of a certain density. That is, the openingsin the mesh material must be sufficiently small to render the constructsutureable or otherwise fastenable, but not so small as to impede properbonding between the foam and the reinforcing mesh as the foam materialand the open cells and cell walls thereof penetrate the mesh openings.Without proper bonding the integrity of the layered structure iscompromised leaving the construct fragile and difficult to handle.Because the density of the mesh determines the mechanical strength ofthe construct, the density of the mesh can vary according to the desireduse for tissue repair. In addition, the type of weave used in the meshcan determine the directionality of the mechanical strength of theconstruct, as well as the mechanical properties of the reinforcingmaterial, such as for example, the elasticity, stiffness, burststrength, suture retention strength and ultimate tensile strength of theconstruct. By way of non-limiting example, the mesh reinforcing materialin a foam-based biocompatible scaffold of the present invention can bedesigned to be stiff in one direction, yet elastic in another, oralternatively, the mesh reinforcing material can be made isotropic.

[0069] During the lyophilization of the reinforced foam, severalparameters and procedures are important to produce implants with thedesired integrity and mechanical properties. Preferably, thereinforcement material is substantially flat when placed in the mold. Toensure the proper degree of flatness, the reinforcement (e.g., mesh) ispressed flat using a heated press prior to its placement within themold. Further, in the event that reinforcing structures are notisotropic it is desirable to indicate this anisotropy by marking theconstruct to indicate directionality. This can be accomplished byembedding one or more indicators, such as dyed markings or dyed threads,within the woven reinforcements. The direction or orientation of theindicator will indicate to a surgeon the dimension of the implant inwhich physical properties are superior.

[0070] As noted above, the manner in which the polymer solution is addedto the mold prior to lyophilization helps contribute to the creation ofa tissue implant with adequate mechanical integrity. Assuming that amesh reinforcing material will be used, and that it will be positionedbetween two thin (e.g., 0.75 mm) shims it should be positioned in asubstantially flat orientation at a desired depth in the mold. Thepolymer solution is poured in a way that allows air bubbles to escapefrom between the layers of the foam component. Preferably, the mold istilted at a desired angle and pouring is effected at a controlled rateto best prevent bubble formation. One of ordinary skill in the art willappreciate that a number of variables will control the tilt angle andpour rate. Generally, the mold should be tilted at an angle of greaterthan about 1 degree to avoid bubble formation. In addition, the rate ofpouring should be slow enough to enable any air bubbles to escape fromthe mold, rather than to be trapped in the mold.

[0071] If a mesh material is used as the reinforcing component, thedensity of the mesh openings is an important factor in the formation ofa resulting tissue implant with the desired mechanical properties. A lowdensity, or open knitted mesh material, is preferred. One preferredmaterial is a 90:10 copolymer of glycolide and lactide, sold under thetradename VICRYL (Ethicon, Inc., Somerville, N.J.). One exemplary lowdensity, open knitted mesh is Knitted VICRYL VKM-M, available fromEthicon, Inc., Somerville, N.J. Other preferred materials arepolydioxanone or 95:5 copolymer of lactide and glycolide.

[0072] The density or “openness” of a mesh material can be evaluatedusing a digital photocamera interfaced with a computer. In oneevaluation, the density of the mesh was determined using a Nikon SMZ-UZoom with a Sony digital photocamera DKC-5000 interfaced with an IBM300PL computer. Digital images of sections of each mesh magnified to 20×were manipulated using Image-Pro Plus 4.0 software in order to determinethe mesh density. Once a digital image was captured by the software, theimage was thresholded such that the area accounting for the empty spacesin the mesh could be subtracted from the total area of the image. Themesh density was taken to be the percentage of the remaining digitalimage. Implants with the most desirable mechanical properties were foundto be those with a mesh density in the range of about 12 to 80% and morepreferably about 45 to 80%.

[0073] In one embodiment, the preferred scaffold for cartilage repair isa mesh reinforced foam. More preferably, the foam is reinforced with amesh that includes polydioxanone (PDO) and the foam composition is acopolymer of 35:65 ε-caprolactone and glycolide. For articularcartilage, the preferred structure to allow cell and tissue ingrowth isone that has an open pore structure and is sized to sufficiently allowcell migration. A suitable pore size is one in which an average diameteris in the range of about 50 to 1000 microns, and more preferably,between about 50 to 500 microns. The mesh layer has a thickness in therange of 1 micron to 1000 microns. Preferably, the foam has a thicknessin the range of about 300 microns to 2 mm, and more preferably, betweenabout 500 microns and 1.5 mm. Preferably, the mesh layer has a meshdensity in the range of about 12 to 80% and more preferably about 45 to80%.

[0074] In another embodiment, the preferred scaffold for cartilagerepair is a nonwoven structure. More preferably, the composition of thenonwoven structure are PANACRYL, a 95:5 copolymer of lactide andglycolide, VICRYL, a 90:10 copolymer of glycolide and lactide, or ablend of polydioxanone and VICRYL sold under the tradename ETHISORB(Johnson & Johnson International, Belgium). For articular cartilage, thepreferred structure to allow cell and tissue ingrowth is one that has anopen pore structure and is sized to sufficiently allow cell migration. Asuitable pore size for the nonwoven scaffold is one in which an averagediameter is in the range of about 50 to 1000 microns and more preferablybetween about 100 to 500 microns. The nonwoven scaffold has a thicknessbetween about 300 microns and 2 mm, and more preferably, between about500 microns and 1.5 mm.

[0075] In one embodiment, the preferred scaffold for meniscus repair isa mesh reinforced foam. More preferably, the foam is reinforced foamwith a mesh that includes polydioxanone (PDO) and the foam compositionis a copolymer of 35:65 ε-caprolactone and glycolide. The preferredstructure to allow cell and tissue ingrowth is one that has an open porestructure and is sized to sufficiently allow cell migration. A suitablepore size is one in which an average diameter is in the range of about50 to 1000 microns, and more preferably, between about 50 to 500microns. The mesh layer has a thickness in the range of about 1 micronto 1000 microns. Preferably, the foam has a thickness in the range ofabout 300 microns to 2 mm, and more preferably, between about 500microns and 1.5 mm. In this embodiment, the preferred method of use isto surround the minced cartilage tissue with this scaffold material.Preferably, the mesh layer has a mesh density in the range of about 12to 80% and more preferably about 45 to 80%.

[0076] In one embodiment, the preferred scaffold for tendon or ligamentrepair is a mesh reinforced foam. More preferably, the foam isreinforced with a mesh that includes polydioxanone (PDO) and the foamcomposition is a copolymer of 35:65 ε-caprolactone and glycolide. Thepreferred structure to allow cell and tissue ingrowth is one that has anopen pore structure and is sized to sufficiently allow cell migration. Asuitable pore size is one in which an average diameter is in the rangeof about 50 to 1000 microns, and more preferably, between about 50 to500 microns. The mesh layer has a thickness in the range of about 1micron to 1000 microns. Preferably, the foam has a thickness in therange of about 300 microns to 2 mm, and more preferably, between about500 microns and 1.5 mm. Preferably, the mesh layer has a mesh density inthe range of about 12 to 80% and more preferably about 45 to 80%.

[0077] In another embodiment, the preferred scaffold for tendon orligament repair is constructed from a polymer that has a slow resorptionprofile (e.g., at least three months, and preferably, at least sixmonths) and high mechanical strength. More preferably, the tensilestrength and elastic modulus of the scaffold must be similar to that ofnative ligament. The preferred tensile strength of the scaffold isbetween about 500N and 4000N, and more preferably, between about 1000Nand 2500N. The preferred elastic modulus of the scaffold is betweenabout 100 N/m and 300 N/m, and more preferably, between about 150 N/mand 200 N/m. The preferred structure of this scaffold is acylindrical-shaped or elliptically-shaped scaffold or a scaffold with ahigh aspect ratio (i.e., ratio of length to width). Preferably, theaspect ratio is greater than 1, and more preferably it is greater than 2and less than 100. Further, the scaffold preferably has a diameter orwidth in the range of about 3 mm and 12 mm, and more preferably, betweenabout 4 mm and 10 mm. By way of non-limiting example, the scaffold forligament repair can include a 95:5 copolymer of lactide and glycolide.In one embodiment, the scaffold for ligament repair can be formed as acomposite structure including a 95:5 copolymer of lactide and glycolideand other polymers, such as for example, polylactide, polyglycolide,polydioxanone, polycaprolactone and combinations thereof. The scaffoldmay be formed of a woven, knit or braided material. Optionally, thepolymers from which the scaffold is made can be formed as a nonwoven,textile structure, such as for example, a weave or a mesh structure, oralternatively these polymers can be formed as a foam. In anotherembodiment, the composite structure can include natural polymers, suchas for example, collagen, fibrin, or silk. In this embodiment, thenatural polymer can act as a coating to the composite structure, oralternatively, the natural polymer can be formed as a foam. Thecomposite structure can also optionally include strips of collagen orsilk to reside within the whole scaffold or just the periphery of thescaffold.

[0078] In one embodiment, the scaffold useful for ligament or tendonrepair is formed of a plurality of filaments, a majority of the fibersof which are aligned in the longitudinal direction.

[0079] One of ordinary skill in the art will appreciate that theselection of a suitable material for forming the biocompatible scaffoldof the present invention depends on several factors. These factorsinclude in vivo mechanical performance; cell response to the material interms of cell attachment, proliferation, migration and differentiation;biocompatibility; and optionally, bioabsorption (or bio-degradation)kinetics. Other relevant factors include the chemical composition,spatial distribution of the constituents, the molecular weight of thepolymer, and the degree of crystallinity.

[0080] In addition to the biocompatible scaffold, the tissue repairimplants of the present invention further include at least one sample ofviable tissue that is associated with at least a portion of thescaffold. The term “viable,” as used herein, refers to a tissue samplehaving one or more viable cells. Virtually any type of tissue can beused to construct the tissue repair implants of the present invention.Preferably, the tissue used is selected from cartilage tissue, meniscaltissue, ligament tissue, tendon tissue, skin tissue, bone tissue, muscletissue, periosteal tissue, pericardial tissue, synovial tissue, nervetissue, fat tissue, kidney tissue, bone marrow, liver tissue, bladdertissue, pancreas tissue, spleen tissue, intervertebral disc tissue,embryonic tissue, periodontal tissue, vascular tissue, blood andcombinations thereof. In one embodiment useful for cartilage repair, thetissue is free of bone tissue and is selected from the group consistingof cartilage tissue, meniscal tissue, ligament tissue and tendon tissue.The tissue used to construct the tissue implant can be autogeneictissue, allogeneic tissue, or xenogeneic tissue.

[0081] In one embodiment useful for meniscal repair, the tissue used inthe tissue repair implant can be selected from the group consisting ofmeniscal tissue, cartilage tissue, skin, synovial tissue, periostealtissue, pericardial tissue, fat tissue, bone marrow, blood, tendontissue, ligament tissue, or combinations thereof. In one embodimentuseful for ligament repair, the tissue used in the tissue repair implantcan be selected from the group consisting of tendon tissue, ligamenttissue of the same type that is to be repaired, ligament tissue of adifferent type than the tissue that is to be repaired, synovial tissue,periosteal tissue, fascia, skin, and combinations thereof.

[0082] The tissue can be obtained using any of a variety of conventionaltechniques, such as for example, by biopsy or other surgical removal.Preferably, the tissue sample is obtained under aseptic conditions. Oncea sample of living tissue has been obtained, the sample can then beprocessed under sterile conditions to create a suspension having atleast one minced, or finely divided, tissue particle. The particle sizeof each tissue fragment can vary, for example, the tissue size can be inthe range of about 0.1 and 3 mm³, in the range of about 0.5 and 1 mm³,in the range of about 1 to 2 mm³, or in the range of about 2 to 3 mm³,but preferably the tissue particle is less than 1 mm³.

[0083] Preferably, the minced tissue has at least one viable cell thatcan migrate from the tissue fragment onto the scaffold. More preferably,the tissue contains an effective amount of cells that can migrate fromthe tissue fragment and begin populating the scaffold. In an optionalembodiment, the minced tissue fragments may be contacted with amatrix-digesting enzyme to facilitate cell migration out of theextracellular matrix surrounding the cells. The enzymes are used toincrease the rate of cell migration out of the extracellular matrix andinto the scaffold material. Suitable matrix-digesting enzymes that canbe used in the present invention include, but are not limited to,collagenase, chondroitinase, trypsin, elastase, hyaluronidase, petidase,thermolysin and protease.

[0084] In one embodiment, the minced tissue particles can be formed as asuspension in which the tissue particles are associated with aphysiological buffering solution. Suitable physiological bufferingsolutions include, but are not limited to, saline, phosphate buffersolution, Hank's balanced salts, Tris buffered saline, Hepes bufferedsaline and combinations thereof. In addition, the tissue can be mincedin any standard cell culture medium known to those having ordinary skillin the art, either in the presence or absence of serum. Prior todepositing the suspension of minced tissue on the scaffold or at thesite of tissue injury, the minced tissue suspension can be filtered andconcentrated, such that only a small quantity of physiological bufferingsolution remains in the suspension to prevent the tissue particles fromdrying out, and the minced tissue particles can be directly applied tothe scaffold or site of injury. Preferably, the minced tissue particlesare loaded at a concentration in the range of approximately 1 to 100mg/cm², and more preferably in the range of about 1 to 20 mg/cm².

[0085] The suspension of minced living tissue can be used to create atissue repair implant according to the present invention by depositingthe suspension of living tissue upon a biocompatible scaffold, such thatthe tissue and the scaffold become associated. Preferably, the tissue isassociated with at least a portion of the scaffold. The tissue repairimplant can be implanted in a subject immediately, or alternatively, theconstruct can be incubated under sterile conditions for a duration andunder conditions that are effective to maintain the viability of thetissue sample. In embodiments where the construct is incubated, theincubation conditions can vary, but preferably, the construct isincubated for a duration in the range of 1 hour to 6 weeks, and morepreferably between about 1 week and 6 weeks, at a temperature in therange of about 20 to 40° C., and in an atmosphere containing betweenabout 5 and 10% carbon dioxide (CO₂) and high humidity, e.g.,approximately 100% humidity.

[0086] A kit can be used to assist in the preparation of the tissuerepair implants of the present invention. According to the presentinvention, the kit includes a sterile container that houses one or morebiocompatible scaffolds, a harvesting tool for collecting the livingtissue sample from a subject, and one or more reagents for sustainingthe viability of the tissue sample. Suitable reagents for sustaining theviability of the tissue sample include a physiological solution, such asfor example, saline, phosphate buffering solution, Hank's balancedsalts, standard cell culture medium, Dulbecco's modified Eagle's medium,ascorbic acid, HEPES, nonessential amino acid, L-proline, fetal bovineserum, autologous serum, and combinations thereof. The kit can alsoinclude a processing tool for dividing the tissue into minced tissueparticles, or alternatively, the harvesting tool can be adapted tocollect the tissue sample and to process the sample into finely dividedtissue particles. The kit can, optionally, also include a deliverydevice for transferring the scaffold from the sterile container to asubject for implantation.

[0087] A biological component may, optionally, be incorporated withinthe tissue repair implants of the present invention. Preferably, thebiological component is incorporated within, or coated on, the scaffoldsdisclosed above. In embodiments where the biological component is coatedonto the scaffold, the biological component is preferably associatedwith at least a portion of the scaffold. By way of nonlimiting example,the biocompatible scaffold can include an adhesion agent for anchoringthe suspension of minced tissue fragments to the scaffold. Preferably,the adhesion agent is an anchoring agent, a cross-linking agent (i.e.,chemical or physical), and combinations thereof.

[0088] Suitable anchoring agents include, but are not limited to,hyaluronic acid, fibrin glue, fibrin clot, collagen gel, alginate gel,gelatin-resorcin-formalin adhesive, mussel-based adhesive,dihydroxyphenylalanine (DOPA) based adhesive, chitosan,transglutaminase, poly(amino acid)-based adhesive, cellulose-basedadhesive, polysaccharide-based adhesive, synthetic acrylate-basedadhesives, platelet rich plasma (PRP), platelet poor plasma (PPP), clotof PRP, clot of PPP, Matrigel, Monostearoyl Glycerol co-Succinate(MGSA), Monostearoyl Glycerol co-Succinate/polyethylene glycol(MGSA/PEG) copolymers, laminin, elastin, proteoglycans, and combinationsthereof.

[0089] Suitable cross-linking agents include, for example, divinylsulfone (DVS), polyethylene glycol divinyl sulfone (VS-PEG-VS),hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA), formaldehyde,glutaraldehyde, aldehydes, isocyanates, alkyl and aryl halides,imidoesters, N-substituted maleimides, acylating compounds,carbodiimide, hydroxychloride, N-hydroxysuccinimide, light (e.g., bluelight and UV light), pH, temperature, and combinations thereof.

[0090] The biological components used in the present invention can alsobe selected from among a variety of effectors that, when present at thesite of injury, promote healing and/or regeneration of the affectedtissue. In addition to being compounds or agents that actually promoteor expedite healing, the effectors may also include compounds or agentsthat prevent infection (e.g., antimicrobial agents and antibiotics),compounds or agents that reduce inflammation (e.g., anti-inflammatoryagents), compounds that prevent or minimize adhesion formnation, such asoxidized regenerated cellulose (e.g., INTERCEED and Surgicel®, availablefrom Ethicon, Inc.), hyaluronic acid, and compounds or agents thatsuppress the immune system (e.g., immunosuppressants).

[0091] By way of example, other types of effectors present within theimplant of the present invention can include heterologous or autologousgrowth factors, proteins (including matrix proteins), peptides,antibodies, enzymes, platelets, glycoproteins, hormones, cytokines,glycosaminoglycans, nucleic acids, analgesics, viruses, virus particles,and cell types. It is understood that one or more effectors of the sameor different functionality may be incorporated within the implant.

[0092] Examples of suitable effectors include the multitude ofheterologous or autologous growth factors known to promote healingand/or regeneration of injured or damaged tissue. These growth factorscan be incorporated directly into the biocompatible scaffold, oralternatively, the biocompatible scaffold can include a source of growthfactors, such as for example, platelets. Exemplary growth factorsinclude, but are not limited to, TGF-β, bone morphogenic protein,cartilage-derived morphogenic protein, fibroblast growth factor,platelet-derived growth factor, vascular endothelial cell-derived growthfactor (VEGF), epidermal growth factor, insulin-like growth factor,hepatocyte growth factor, and fragments thereof. Suitable effectorslikewise include the agonists and antagonists of the agents noted above.The growth factor can also include combinations of the growth factorslisted above. In addition, the growth factor can be autologous growthfactor that is supplied by platelets in the blood. In this case, thegrowth factor from platelets will be an undefined cocktail of variousgrowth factors.

[0093] The proteins that may be present within the implant includeproteins that are secreted from a cell or other biological source, suchas for example, a platelet, which is housed within the implant, as wellas those that are present within the implant in an isolated form. Theisolated form of a protein typically is one that is about 55% or greaterin purity, i.e., isolated from other cellular proteins, molecules,debris, etc. More preferably, the isolated protein is one that is atleast 65% pure, and most preferably one that is at least about 75 to 95%pure. Notwithstanding the above, one of ordinary skill in the art willappreciate that proteins having a purity below about 55% are stillconsidered to be within the scope of this invention. As used herein, theterm “protein” embraces glycoproteins, lipoproteins, proteoglycans,peptides, and fragments thereof. Examples of proteins useful aseffectors include, but are not limited to, pleiotrophin, endothelin,tenascin, fibronectin, fibrinogen, vitronectin, V-CAM, I-CAM, N-CAM,selectin, cadherin, integrin, laminin, actin, myosin, collagen,microfilament, intermediate filament, antibody, elastin, fibrillin, andfragments thereof.

[0094] Glycosaminoglycans, highly charged polysaccharides which play arole in cellular adhesion, may also serve as effectors according to thepresent invention. Exemplary glycosaminoglycans useful as effectorsinclude, but are not limited to, heparan sulfate, heparin, chondroitinsulfate, dermatan sulfate, keratan sulfate, hyaluronan (also known ashyaluronic acid), and combinations thereof.

[0095] The biocompatible scaffolds of the present invention can alsohave cells incorporated therein. Suitable cell types that can serve aseffectors according to this invention include, but are not limited to,osteocytes, osteoblasts, osteoclasts, fibroblasts, stem cells,pluripotent cells, chondrocyte progenitors, chondrocytes, endothelialcells, macrophages, leukocytes, adipocytes, monocytes, plasma cells,mast cells, umbilical cord cells, stromal cells, mesenchymal stem cells,epithelial cells, myoblasts, tenocytes, ligament fibroblasts, neurons,and bone marrow cells. Cells typically have at their surface receptormolecules which are responsive to a cognate ligand (e.g., a stimulator).A stimulator is a ligand which when in contact with its cognate receptorinduce the cell possessing the receptor to produce a specific biologicalaction. For example, in response to a stimulator (or ligand) a cell mayproduce significant levels of secondary messengers, like Ca⁺², whichthen will have subsequent effects upon cellular processes such as thephosphorylation of proteins, such as (keeping with our example) proteinkinase C. In some instances, once a cell is stimulated with the properstimulator, the cell secretes a cellular messenger usually in the formof a protein (including glycoproteins, proteoglycans, and lipoproteins).This cellular messenger can be an antibody (e.g., secreted from plasmacells), a hormone, (e.g., a paracrine, autocrine, or exocrine hormone),a cytokine, or natural or synthetic fragments thereof.

[0096] The tissue implants of the invention can also be used in genetherapy techniques in which nucleic acids, viruses, or virus particlesdeliver a gene of interest, which encodes at least one gene product ofinterest, to specific cells or cell types. Accordingly, the biologicaleffector can be a nucleic acid (e.g., DNA, RNA, or an oligonucleotide),a virus, a virus particle, or a non-viral vector. The viruses and virusparticles may be, or may be derived from, DNA or RNA viruses. The geneproduct of interest is preferably selected from the group consisting ofproteins, polypeptides, interference ribonucleic acids (iRNA) andcombinations thereof.

[0097] Once the applicable nucleic acids and/or viral agents (i.e.,viruses or viral particles) are incorporated into the biocompatiblescaffold of the tissue repair implant, the implant can then be implantedinto a particular site to elicit a type of biological response. Thenucleic acid or viral agent can then be taken up by the cells and anyproteins that they encode can be produced locally by the cells. In oneembodiment, the nucleic acid or viral agent can be taken up by the cellswithin the tissue fragment of the minced tissue suspension, or, in analternative embodiment, the nucleic acid or viral agent can be taken upby the cells in the tissue surrounding the site of the injured tissue.One of ordinary skill in the art will recognize that the proteinproduced can be a protein of the type noted above, or a similar proteinthat facilitates an enhanced capacity of the tissue to heal an injury ora disease, combat an infection, or reduce an inflammatory response.Nucleic acids can also be used to block the expression of unwanted geneproduct that may impact negatively on a tissue repair process or othernormal biological processes. DNA, RNA and viral agents are often used toaccomplish such an expression blocking function, which is also known asgene expression knock out.

[0098] One of ordinary skill in the art will appreciate that theidentity of the biological component may be determined by a surgeon,based on principles of medical science and the applicable treatmentobjectives.

[0099] The biological component or effector of the issue repair implantcan be incorporated within the scaffold before or after manufacture ofthe scaffold, or before or after the surgical placement of the implant.

[0100] Prior to surgical placement, the biocompatible scaffold can beplaced in a suitable container comprising the biological component.After an appropriate time and under suitable conditions, the scaffoldwill become impregnated with the biological component. Alternatively,the biological component can be incorporated within the scaffold by, forexample, using an appropriately gauged syringe to inject the biologicalagent(s) into the scaffold. Other methods well known to those ofordinary skill in the art can be applied in order to load a scaffoldwith an appropriate biological component, such as mixing, pressing,spreading, centrifuging and placing the biological component into thescaffold. Alternatively, the biological component can be mixed with agel-like carrier prior to injection into the scaffold. The gel-likecarrier can be a biological or synthetic hydrogel, including analginate, a cross-linked alginate, hyaluronic acid, collagen gel,poly(N-isopropylacrylamide), poly(oxyalkylene), a copolymer ofpoly(ethylene oxide)-poly(propylene oxide), poly(vinyl alcohol) andblends thereof.

[0101] Following surgical placement, an implant wherein thebiocompatible scaffold is devoid of any biological component can beinfused with biological agent(s), or an implant wherein the scaffoldincludes at least one biological component can be augmented with asupplemental quantity of the biological component. One method ofincorporating a biological component within a surgically installedimplant is by injection using an appropriately gauged syringe.

[0102] The amount of the biological component included with abiocompatible scaffold will vary depending on a variety of factors,including the size of the scaffold, the material from which the scaffoldis made, the porosity of the scaffold, the identity of the biologicallycomponent, and the intended purpose of the tissue repair implant. One ofordinary skill in the art can readily determine the appropriate quantityof biological component to include within a biocompatible scaffold for agiven application in order to facilitate and/or expedite the healing oftissue. The amount of biological component will, of course, varydepending upon the identity of the biological component and the givenapplication.

[0103] In another embodiment, the tissue repair implant can include anadditional retaining element that is placed over the tissue-ladenscaffold. Preferably, in this embodiment, at least a portion of thetissue suspension is associated with at least a portion of the outersurface of the scaffold, such that the tissue suspension is “sandwiched”between the biocompatible scaffold and the retaining element. Theretaining element can be formed from virtually any biocompatiblematerial, and in one embodiment, the retaining element can be formedusing tissue grafts, including grafts obtained from allogeneic tissue,autogeneic tissue, and xenogeneic tissue, an additional biocompatiblescaffold selected from the biocompatible scaffolds disclosed above, andcombinations thereof. In another embodiment, the retaining element canbe a porous mesh, a porous mesh-like material, such as for example, aknit, a weave, a nonwoven, or a thin, perforated elastomeric sheethaving pores or perforations to allow tissue ingrowth. The thin,perforated elastomeric sheets are preferably constructed from collagenor silk or blends or copolymers of polylactic acid (PLA), polyglycolicacid (PGA), polycaprolactone (PCL) and polydioxanone (PDO). The type ofretaining element used can vary according to the desired tissue repair.By way of non-limiting example, in one embodiment for meniscus repair,the retaining element can be a mesh-reinforced foam. In embodiments forACL and cartilage repair, the retaining element can be a mesh structure.In embodiments where the retaining element is an allograft or anautograft, preferably the allograft or autograft is selected fromperiosteum, perichondrium, iliotibial band or fascia lata, gracilistendon, semitendinosis tendon, patellar tendon, synovium andcombinations thereof. In embodiments where the retaining element is axenograft, the xenograft is preferably selected from the correspondinganatomical structure for small intestine, periosteum, perichondrium,iliotibial band or fascia lata, gracilis tendon, semitendonous tendon,patellar tendon, synovium, and combinations thereof. These retainingelements can be placed over the biocompatible scaffold, oralternatively, the retaining element can be affixed, such as forexample, by suturing or stapling, the implant to act as a retainingelement. One of ordinary skill in the art will appreciate thatadditional processing of the retaining element, such as for example, theplacement of holes within the retaining element, may be determined by asurgeon, based on principles of medical science and the applicabletreatment objectives.

[0104] In yet another embodiment, an electrostatically spun fabricbarrier may be added to the implant to act as a barrier to hyperplasiaand tissue adhesion, thus reducing the possibility of postsurgicaladhesions. The fabric barrier is preferably in the form of dense fibrousfabric that is added to the implant. Preferably, the fibrous fabric iscomprised of small diameter fibers that are fused to the top and/orbottom surface of the biocompatible scaffold. This enables certainsurface properties of the structure, such as porosity, permeability,degradation rate and mechanical properties, to be controlled.

[0105] One of ordinary skill in the art will appreciate that the fibrousfabric can be produced via an electrostatic spinning process in which afibrous layer can be built up on lyophilized foam and nonwoven surfaces.This electrostatic spinning process may be conducted using a variety offiber materials. Exemplary fiber materials include aliphatic polyesters.A variety of solvents may be used as well, including those identifiedabove that are useful to prepare the polymer solution that forms thefoam component.

[0106] The composition, thickness, and porosity of the fibrous layer maybe controlled to provide the desired mechanical and biologicalcharacteristics. For example, the bioabsorption rate of the fibrouslayer may be selected to provide a longer or shorter bioabsorptionprofile as compared to the underlying biocompatible scaffold.Additionally, the fibrous layer may provide greater structural integrityto the composite so that mechanical force may be applied to the fibrousside of the structure. In one embodiment the fibrous layer could allowthe use of sutures, staples or various fixation devices to hold thecomposite in place. Generally, the fibrous layer has a thickness in therange of about 1 micron to 1000 microns. However, for some applicationssuch as rotator cuff and meniscus injury repair, the fibrous layer has athickness greater than about 1.5 mm.

[0107] The tissue repair implants of the present invention can be usedin a variety of surgical and non-surgical applications. In some surgicalapplications, such as for use in the repair of a variety of tissuesincluding a torn ligament, tendon, rotator cuff, nerve, skin, cartilage,or meniscus, the tissue implants of the invention must be able to behandled in the operating room, and they must be able to be sutured orotherwise fastened without tearing. Additionally, the implants shouldhave a burst strength adequate to reinforce the tissue, and thestructure of the implant can be suitable to encourage tissue ingrowth.By way of non-limiting example, the scaffolds of the present inventioncan be highly porous to allow cell growth therein. Preferably, themedian pore size is in the range of about 100 to 500 microns. In theseembodiments, the scaffold should be sufficiently pliable to accommodatetissue growth within the interior region of the scaffold, so that thegeometry of the scaffold can be remodeled as tissue ingrowth increases.Accordingly, in the present invention, tissue can be grown on thesurface of the biocompatible scaffold, or alternatively, tissue can begrown into and on the surface of the biocompatible scaffold, such thatthe tissue becomes embedded in and integrated with the scaffold.

[0108] In one embodiment of the present invention, the tissue repairimplant is used in the treatment of a tissue injury, such as injury to aligament, tendon, nerve, skin, cartilage or meniscus. Repairing tissueinjuries involves the steps of obtaining a sample of living tissue byany of the variety of techniques known to those having ordinary skill inthe art, processing that sample of living tissue under sterileconditions, such as for example by cutting the tissue, to create atleast one minced, finely divided tissue particle, depositing the tissuesample upon the biocompatible scaffold, such that the tissue samplebecomes associated with the scaffold to form a tissue repair implant,and placing the tissue repair implant in a desired position relative tothe tissue injury. Repairing tissue injuries may also involve placingthe scaffold at the site of tissue injury and then depositing the finetissue particles onto the scaffold. The cells in the tissue particlesassociated with the scaffold can migrate to the scaffold and beginproliferating and integrating with surrounding tissue at the site ofimplantation, thereby repairing the tissue injury. This method forrepairing tissue injuries can include an additional, optional step.Prior to the step of placing the tissue repair implant in a desiredposition relative to the tissue injury, the scaffold and associatedtissue particles can be incubated for a duration and under conditionseffective to allow cells within the tissue particles to migrate from thetissue and begin populating the scaffold.

[0109] The tissue samples used in the present invention are obtainedfrom a donor (autogenic, allogeneic, or xenogeneic) using appropriateharvesting tools. The tissue samples can be finely minced and dividedinto small particles either as the tissue is collected, oralternatively, the tissue sample can be minced after it is harvested andcollected outside the body. In embodiments, where the tissue sample isminced after it is harvested, the tissue samples can be weighed and thenwashed three times in phosphate buffered saline. Approximately 300 to500 mg of tissue can then be minced in the presence of a small quantity,such as, for example, about 1 ml, of a physiological buffering solution,such as, for example, phosphate buffered saline, or a matrix digestingenzyme, such as, for example, 0.2% collagenase in Hams F12. Mincing thetissue divides the tissue into particles or small pieces ofapproximately 1 mm³. Mincing the tissue can be accomplished by a varietyof methods. In one embodiment, the mincing is accomplished with twosterile scalpels using a parallel direction, and in another embodiment,the tissue can be minced by a processing tool that automatically dividesthe tissue into particles of a desired size. In one embodiment, theminced tissue can be separated from the physiological fluid andconcentrated using any of a variety of methods known to those havingordinary skill in the art, such as for example, sieving, sedimenting orcentrifuging. In embodiments where the minced tissue is filtered andconcentrated, the suspension of minced tissue preferably retains a smallquantity of fluid in the suspension to prevent the tissue from dryingout. In another embodiment, the suspension of minced tissue is notconcentrated, and the minced tissue can be directly delivered to thesite of tissue repair via a high concentration tissue suspension orother carrier such as for example, a hydrogel, fibrin glue, or collagen.In this embodiment, the minced tissue suspension can be covered by anyof the biocompatible scaffolds described above to retain the tissuefragments in place.

[0110] The minced tissue can then be distributed onto a scaffold using acell spreader so as to cover the entire scaffold. In a preferableembodiment for meniscus and cartilage repair, the minced tissue isspread onto 4×5 cm scaffolds that have been presoaked in Dulbecco'smodified Eagles medium (DMEM) so as to cover the entire scaffold.Optionally, the tissue particles can be adhered to the scaffolds usingany of the adhesive agents described above, such as, for example, fibringlue or platelet rich plasma. In embodiments using fibrin glue orplatelet rich plasma, a few microliters of thrombin can be placed on thescaffolds, prior to distribution of fibrinogen or platelet rich plasmaon the scaffolds, and allowed to set. Once the tissue particles and anyadditional agents have been deposited on the scaffold, the tissue repairimplant can then implanted immediately, or alternatively, the implantcan be cultured in vitro for a duration and under conditions sufficientto allow the cells in the tissue particles to migrate from the tissueparticles onto the scaffold. In an embodiment where the tissue repairimplant is incubated prior to implantation, the implant is preferablycultured in vitro for approximately 1-3 weeks in a chondrocyte growthmedium, such as for example, DMEM-high glucose, supplemented with 20%fetal calf serum (FCS), 10 mM HEPES, 0.1 mM nonessential amino acids, 20mg/ml of L-proline, 50 mg/ml ascorbic acid, 100 mg/ml penicillin, 100mg/ml of streptomycin and 0.25 mg/ml of amphotericin B.

[0111] The methods of repairing tissue injuries using the tissueimplants according to the present invention can be conducted during asurgical operation to repair the tissue injury. Alternatively, the stepsof processing the tissue sample to create minced, finely divided tissueparticles, depositing the tissue particles upon the scaffold to form atissue repair implant, and/or incubating the tissue repair implant priorto implantation can be conducted at another, sterile location prior tosurgical placement of the implant relative to the site of injury.

[0112] The implants used to repair injured tissue can be of a size andshape such that they match the geometry and dimensions of a desiredportion or lesion of the tissue to be treated. The implant can be sizedand shaped to produce the necessary geometry by numerous techniquesincluding cutting, folding, rolling, or otherwise manipulating theimplant. As noted above, the biological component may be added to thescaffold during or after manufacture of the scaffold or before or afterthe implant is installed in a patient. An additional quantity of thebiological component may be added after the implant is installed. Onceaccess is made into the affected anatomical site (whether by minimallyinvasive, open or mini-open surgical technique), the implant can beaffixed to a desired position relative to the tissue injury, such aswithin a tear or lesion. Once the implant is placed in the desiredposition or lesion, it can be affixed by using a suitable technique. Inone aspect, the implant can be affixed by a chemical and/or mechanicalfastening technique. Suitable chemical fasteners include glues and/oradhesive such as fibrin glue, fibrin clot, and other known biologicallycompatible adhesives. Suitable mechanical fasteners include sutures,staples, tissue tacks, suture anchors, darts, screws, pins and arrows.It is understood that combinations of one or more chemical and/ormechanical fasteners can be used. Alternatively, one need not use anychemical and/or mechanical fasteners. Instead, placement of the implantcan be accomplished through an interference fit of the implant with anappropriate site in the tissue to be treated.

[0113] In another embodiment, the tissue repair implant is useful insurgical techniques that repair ligaments, tendons, skin and/or nerves.

[0114] In one use, the tissue repair implant can be for repair and toaugment tissue loss during tendon or ligament repair surgery or it canbe used as a stand alone device. In the case of repair, tendon orligament ends are approximated through appropriate surgical techniquesand the tissue repair implant is used around the joined end to give moremechanical support and to enhance the healing response. As a result ofthe healing process, the tendon or ligament tissue grows within theimplant device, eventually maturing into a tissue with similarmechanical properties to that of native tissue. The implant provides themechanical support that is initially necessary to ensure proper healing,and it also serves as a guide for tissue regeneration. In another use asa stand alone device, the ruptured tissue is removed, and the tissuerepair implant with minced tissue serves to replace the function of thedamaged tissue. The ruptured tissue can be the tissue source used forhealing damaged tissue.

[0115] In embodiments where the tissue repair implant is used to repairligament tissue, the tissue repair implant can be used for tissueaugmentation, or alternatively, as a stand-alone device. In embodimentswhere the tissue repair implant is used for augmentation, the tissuerepair implant can be used in conjunction with any of a variety ofstandard, established repair techniques known to those having ordinaryskill in the art. In embodiments where the tissue repair implant is usedfor augmentation during ACL repair, surgeons currently use an autograftconsisting of ligament tissue, bone-patellar tendons, tendon-bonetendons, hamstring tendons, or iliotibial band to repair tissue, and thetissue repair implant of the present invention can be placed eitheraround the autograft, surrounded by the autograft, or alongside theautograft. In embodiments where the tissue repair element is used as astand-alone device, the ruptured ligament can be removed and completelyreplaced by the tissue repair implant. In this case, the tissue repairimplant can be affixed to bone at each end of the implant. In the caseof ACL repair, one end of the implant can be stabilized at the originalorigin site of the femur, while the other end can be placed at theoriginal insertion site on the tibia.

[0116] The tissue repair implant can be utilized in a variety ofconfigurations. For example, the implant can be folded or stacked inmultiple laminates or it can be rolled into the shape or a tube-likestructure. Tendon or ligament ends can be joined, for example, bysuturing, stapling, clipping, adhering, or anchoring, the implant toends of the implant. In some embodiments where the tissue repair implantis used to repair tendons, such as for example, rotator cuff repair, thesurgeon can use the tissue repair implant to assist in thereapproximation of the torn rotator cuff to a bony trough through thecortical surface of the greater tuberosity. Often times, in olderpatients, the rotator cuff tissue is thin and degenerate and/or thequality of the humerus is osteoporotic. Therefore, in order to increasethe strength of the attachment to the bony trough, the tissue repairimplant can be placed on top of the tendon, such that the sutures wouldpass through both the scaffold and tendon, or alternatively, the tissuerepair implant can be used on top of the bone bridge to prevent thesutures from pulling out of the bone. In either embodiment, the tissuerepair implant provides suture retention strength. In situations wherethe quality of the rotator cuff is so degenerate that the tissue cannotbe reapproximated to the humerus, the tissue repair implant can serve asa bridge, wherein one end of the implant can be joined to the remainingtendon while the other end can be attached to the bone.

[0117] In another variation, the implant can be used to repair orreplace the sheath of a tendon. To do so, the implant is sutured orotherwise joined to the connective tissue, such as the periosteum,synovium, or muscle, and wrapped around the tendon. This constructionallows free gliding of the tendon within the sheath formed by theimplant. The implant provides the necessary structural support followingsurgery. Over time, however, the implant in this embodiment can beresorbed and replaced by new tissue.

[0118] The implants of the invention can also be used for organ repairreplacement or regeneration strategies that may benefit from theseunique tissue implants. For example, these implants can be used forspinal disc, cranial tissue, dura, nerve tissue, liver, pancreas,kidney, bladder, uterus, esophagus, liver spleen, cardiac muscle,skeletal muscle, skin, fascia, pelvic floor, stomach, tendons,cartilage, ligaments, and breast tissues.

[0119] In yet another embodiment, the implants of the present inventioncan be used to create a biological assay for measuring the effect of asubstance on living tissue. In this embodiment, tissue constructs arecreated, as described above, by providing a sterile, biocompatiblescaffold, obtaining a sample of living tissue, processing the sample ofliving tissue under sterile conditions to form a suspension of mincedtissue having minced tissue fragments and a physiological bufferingsolution, and depositing the suspension of minced tissue on thebiocompatible scaffold such that the suspension of minced tissue and thescaffold become associated. The tissue construct is incubated underconditions that are effective to allow cells within the minced tissue topopulate the scaffold. The tissue construct can then be contacted withthe substance that is to be tested, and the effect(s) of that substancecan be determined. These tissue constructs can be used to determineand/or test the biological responses to a test substance, such as forexample, cell viability, growth, migration, differentiation andmaintenance of cell phenotype, metabolic activity, induction orrepression. These biological responses can be assayed using any of avariety of techniques known to those having ordinary skill in the art,such as for example, proliferation assay, cell migration assay, proteinassay, gene expression assay, viability assay, calorimetric assay ormetabolic assay. By way of non-limiting example, the expression of aselected gene(s) or gene products typically expressed by the tissue ofthe tissue construct, such as for example, the expression of type II,type IX or type XI collagen expressed by chondrocytes, using a varietyknown assays, such as for example, northern blot analysis, RNAseprotection assays, polymerase chain reaction (PCR), western blotanalysis and enzyme-linked immunoabsorbant assay (ELISA). Suitablesubstances that can be tested using the tissue constructs of the presentinvention include, but are not limited to, drugs, pharmaceuticalcompositions, chemicals, microbes, elements, cytokines, growth factors,hormones, antibodies, peptides, ligands, antagonists of membrane-boundreceptors, and combinations thereof.

[0120] The implants of the present invention can also be used as adelivery device for a therapeutic, wherein the therapeutic is the mincedtissue, which includes a combination of cells, extracellular matrix andinherent growth factors. The scaffold portion of the implant can allowfor hormones and proteins to be released into the surroundingenvironment.

[0121] The methods of repairing tissue injuries using the tissueimplants according to the present invention can be conducted during asurgical operation to repair the tissue injury. A patient is preparedfor tissue repair surgery in a conventional manner using conventionalsurgical techniques. Tissue repair is performed at the site of injuredtissue using the tissue repair implants of the present invention. Thetissue sample used to form the tissue repair implant is obtained fromthe patient (or another donor) using appropriate tools and techniques.The tissue sample is finely minced and divided into at least one tissueparticle having a particle size in the range of about 0.1 to 3 mm³. Thetissue can be minced using a conventional mincing technique such as twosterile scalpels used in a parallel direction. Between about 300 to 500mg of tissue is minced in the presence of about 1 ml of a physiologicalbuffering solution, depending on the extent of the tissue injury at thesite of repair. The minced tissue is filtered and concentrated toseparate the minced tissue particle from the physiological bufferingsolution. The minced tissue can be concentrated using any of a varietyof conventional techniques, such as for example, sieving, sedimenting orcentrifuging. The minced tissue particles are then distributed using acell spreader onto a 4×5 cm biocompatible scaffold that has been soakedin Dulbecco's modified Eagles medium (DMEM). An adhesion agent can beadded to the biocompatible scaffold and the minced tissue particles. Thetissue repair implant is implanted at the site of tissue injury, eitherimmediately or after a period of in vitro incubation. Final woundclosure is performed in a conventional manner using conventionalsurgical techniques.

[0122] The following examples are illustrative of the principles andpractice of this invention. Numerous additional embodiments within thescope and spirit of the invention will become apparent to those skilledin the art.

EXAMPLE 1

[0123] Healthy cartilage tissue from articulating joints was obtainedfrom bovine shoulders. The cartilage tissue, which was substantiallyfree of bone tissue, was minced using scalpel blades to obtain smalltissue fragments in the presence of 0.2% collagenase. The size of thetissue fragments varied but on average should be approximately 1×1 mm indimension. The minced tissue was then distributed uniformly on a 4×5 cmsynthetic bioresorbable polycaprolactone/polyglycolic acid (PCL/PGA)scaffold. Ethylene oxide sterilized polymer scaffolds, were pre-soakedfor 4 hours in Dulbecco's Modified Eagle's Medium prior to distributionof tissue fragments. The scaffold loaded with minced fragments was thenplaced in a 10 cm cell culture dish containing chondrocyte growthmedium. The chondrocyte growth medium consisted of Dulbecco's modifiedeagles medium (DMEM-high glucose) supplemented with 20% fetal calf serum(FCS), 10 mM HEPES, 0.1 mM nonessential amino acids, 20 mg/ml ofL-proline, 50 mg/ml ascorbic acid, 100 mg/ml penicillin, 100 mg/ml ofstreptomycin and 0.25 mg/ml of amphotericin B. The growth medium wasreplenished every other day. Scaffolds were cultured at 37° C. in a cellculture incubator. Six weeks following culture samples were removed andanalyzed for cell distribution and migration within the scaffolds andfor production of cartilage like matrix. FIG. 1 demonstrates that cellsmigrate extensively into the polymer scaffolds from the minced cartilagetissue fragments (FIG. 1A). The migrating cells retain their phenotypeand produce matrix that stained positive for the sulfatedglycosaminoglycans using the Safranin O stain (FIG. 1B).

EXAMPLE 2

[0124] The bioresorbable scaffolds containing minced cartilage tissueand cells from Example 1 were also implanted into SCID mice. Theobjective was to evaluate the chondrocytic ingrowth of mincedcartilaginous tissues into polymer scaffolds in vivo. Polymer scaffolds5 mm in diameter, were subcutaneously implanted bilaterally in thelateral thoracic region of SCID mice. The implanted scaffold waspermitted to support cell growth for four weeks. The subcutaneousimplantation sites with their overlying skin were then excised andpreserved in 10% buffered formalin fixative. Following fixation, eachimplantation site was processed for histology. Histological sectionswere stained with Hematoxylin and eosin, and Safranin-O. FIGS. 2 A and Bshow that abundant cells were distributed within the scaffold. The cellsdisplayed chondrocyte-like morphology, as evidenced by the intensepositive staining for Safranin O of the synthesized matrix.

EXAMPLE 3

[0125] Minced cartilage tissue prepared according to the methoddescribed in Example 1 was distributed uniformly on a 4×5 cm syntheticbioresorbable polycaprolactone/polyglycolic acid (PCL/PGA) scaffold.Minced cartilage tissue fragments were adhered to the scaffolds with 1mL of platelet rich plasma (PRP, Human). Sixty microliters (60 units) ofthrombin were used to induce clot formation in the PRP. Controlscaffolds loaded with minced cartilage fragments alone and scaffoldsloaded with minced cartilage fragments adhered by PRP, were cultured invitro for 1 week, and then implanted into SCID mice as described in theExample 2. FIG. 3A is a photomicrograph of a control scaffold loadedwith minced tissue. FIG. 3B is a photomicrograph depicting a scaffoldloaded with minced tissue and PRP. FIG. 3B demonstrates that PRP isbeneficial in promoting the migration of the chondrocyte cells, and PRPis also beneficial in promoting the maintenance of the differentiatedphenotype of the chondrocyte cells within the scaffolds. The migratingcells retain their phenotype and produce matrix that stained positivefor the sulfated glycosaminoglycans using the Safranin O stain (FIG.3B).

EXAMPLE 4

[0126] Healthy full-thickness skin samples, collected from 1×1 cm woundscreated on the dorsal side of the pigs, were immediately placed in 50 mlconical tubes containing DMEM with 10× antibiotics/antimycotics. Tissuesamples were rinsed once in PBS containing 10× antibiotic/antimycoticsfollowed by an additional rinsing step with PBS containing 1×antibiotics/antimycotics. The tissue was minced aseptically using ascalpel blade in a laminar flow hood. Dispersed skin samples weresubjected to enzymatic digestion with 1 ml of 0.25% collagenase/0.25%dispase at 37° C. for 15 min (Autologous cell dispersion #1). Anotherset of samples were first digested with 500 μl of 0.25% trypsin for 10min, then washed with PBS to remove trypsin, and then incubated with 1ml of 0.25% collagenase/0.25% dispase at 37° C. for 15 min (Autologouscell dispersion #2). Following digestion, the samples were centrifugedat 2500 rpm for 5 min. The supernatant was aspirated and discarded.Dispersed, partially digested skin samples were washed once in PBS andthen re-suspended in 500 μl of PBS. Approximately 20 μl of cellsuspension was distributed evenly in the wound bed and bioresorbablescaffold was carefully applied on the top of dispersed cells making surenot to dislodge the cell suspension. Dispersed cells could bedistributed evenly on the scaffold and placed onto the wound bed. FIG. 4demonstrates that autologous cell dispersion was present histologicallyas keratinocyte “islands,” some of which had migrated throughout thescaffold towards the wound surface.

EXAMPLE 5

[0127] Healthy anterior cruciate ligament tissue was obtained frombovine knees. The ligament tissue was minced using scalpel blades and/orscissors to obtain small tissue fragments. While the size of the tissuefragments varied, the average particle size was approximately 1 mm³ indimension. In this example, the ligament was minced with and without0.2% collagenase. The minced tissue was then distributed uniformly on a4×5 cm synthetic bioresorbable polycaprolactone/polyglycolic acidPGA/PCL scaffold or polylactic acid/polyglycolic acid (PLA/PGA)scaffold. The scaffolds were sterilized in 70% ethanol for our hour andwashed three times with sterile PBS. The scaffolds were then pre-soakedfor 1-2 hours in Dulbecco's Modified Eagle's Medium with1×antibiotic-antimycotic prior to distribution of tissue fragments. Thescaffold loaded with minced fragments was then placed in a 10 cm cellculture dish containing growth medium, which consisted of Dulbecco'smodified eagles medium (DMEM-high glucose) supplemented with 20% fetalcalf serum (FCS), 100 mg/ml penicillin, 100 mg/ml of streptomycin and0.25 mg/ml of amphotericin B. Scaffolds with the minced tissue werecultured at 37° C. in a cell culture incubator and the growth medium wasexchanged every other day. Three and six weeks following culture,samples were removed and analyzed for cell distribution and migrationwithin the scaffolds. FIG. 5 demonstrates cells migrating extensivelyinto the polymer scaffolds after 6 weeks in culture from the mincedanterior cruciate tissue fragments treated with collagenase (FIG. 5A)and without collagenase (FIG. 5B).

EXAMPLE 6

[0128] Menisci were harvested from adult Goat knees and 4 mm diameterexplants (2 mm thick) were taken from the white and red/white regions. A2 mm punch biopsy was removed from the center of the explants. Abioresorbable scaffold polylactic acid/polycaprolactone (PLA/PCL) 2 mmin diameter and 2 mm thick was inserted into the center of the meniscalexplant. The explants with scaffolds were cultured for 2 and 3 weeksunder standard cell culture conditions with changes in media (DMEMcontaining 1% FBS, 1×antibiotic-antimycotic) occurring every other day.At 14 and 21 days following culture, half the samples were placed into10% buffered formalin for histological processing. Sections were stainedwith Hematoxylin to visualize the cells. From the remaining samples thescaffolds were removed and cell number estimated by quantitation of DNAusing the CyQuant assay. FIG. 6A demonstrates that there is cellmigration into the polymer scaffolds from the meniscal explants. FIG. 6Bshows the histology of cross sections of scaffolds demonstrating cellmigration into scaffolds.

EXAMPLE 7

[0129] Healthy cartilage tissue and osteochondral plugs were obtainedfrom articulating joints of bovine shoulders. Minced cartilage tissuewas prepared according to the method described in Example 1. Inaddition, osteochondral plugs (1×1 cm) were harvested from bovineshoulders using a diamond bone saw and morselized with bone cutters toobtain bone cartilage paste. Next, 250 mg of the sample (mincedcartilage or bone cartilage paste) was distributed on 2×5 cm syntheticbioresorbable (PCL/PGA) scaffolds. The scaffold loaded with mincedcartilage fragments or osteochondral paste was then placed in a 10 cmcell culture dish containing chondrocyte growth medium and cultured in acell culture incubator as described in Example 1. Three weeks followingculture the samples were removed and implanted into SCID mice asdescribed in Example 2. The objective was to evaluate the nature oftissue formed within the scaffold following implantation for 4 weeks.Histological sections were analyzed for cell distribution and for thenature of the matrix formed, within the scaffolds, by staining withHematoxylin and eosin (H/E), Safranin O (SO) and Modified Mallory'sAniline Blue (MMAB). FIGS. 7A-7C demonstrate that cells migrateextensively into the polymer scaffolds from the minced cartilage tissuefragments and form cartilage like matrix that stains positive forSafranin O. This is particularly evident in FIG. 7B in which the darkerarea in the center and top of the photograph is indicative of positivestaining. FIGS. 8A-8C demonstrate that cells migrate from bone cartilagepaste into polymer scaffolds. However, the tissue that is formedcomprises cartilage as well as new bone. The appearance of the new boneis indicated by the lighter arrows in FIG. 8C while the old bonefragments are indicated by the darker arrows in FIGS. 8 A and 8C.

EXAMPLE 8

[0130] Healthy cartilage tissue was obtained from articulating joints ofbovine shoulders. Minced cartilage tissue was prepared according to themethod described in Example 1. Biopsy punches were used to obtaincartilage tissue fragments 2 mm and 3 mm in diameter. The thickness ofthese fragments was approximately 1 mm. 250 mg of minced cartilage orcartilage fragments 2 or 3 mm in diameter were distributed on 2×5 cmsynthetic bioresorbable (PCL/PGA) scaffold. The scaffold loaded withcartilage fragments was then placed in a 10 cm cell culture dishcontaining chondrocyte growth medium and cultured in a cell cultureincubator as described in Example 1. Three weeks following culturesamples were removed and cell number estimated by quantitation of DNAcontent. 5 mm biopsy punches were also implanted into SCID mice asdescribed in Example 2. The objective was to evaluate the optimal sizeof tissue fragments for this process. FIG. 9 demonstrates that thehighest cell number was observed in scaffolds loaded with mincedcartilage tissue and the lowest in scaffolds loaded with cartilagefragments 3 mm in diameter. FIGS. 10A-10C provide histologicalevaluations of scaffolds implanted into SCID mice and stained withSafranin O. These results demonstrate that uniform cartilage-like tissue(stained, darker areas) in scaffolds loaded with minced cartilage tissueand cartilage fragments 2 mm in diameter (FIGS. 10A and B). Scaffoldsthat were loaded with cartilage fragments 3 mm in diameter were notuniformly filled (FIG. 10C).

[0131] One of ordinary skill in the art will appreciate further featuresand advantages of the invention based on the above-describedembodiments. Accordingly, the invention is not to be limited by what hasbeen particularly shown and described, except as indicated by theappended claims. All publications and references cited herein areexpressly incorporated herein by reference in their entirety.

What is claimed is:
 1. A biocompatible implant, comprising: abiocompatible scaffold; and at least one tissue fragment that isassociated with at least a portion of the scaffold, wherein the tissuefragment includes an effective amount of viable cells that can migrateout of the tissue fragment and populate the scaffold.
 2. The implant ofclaim 1, wherein the scaffold comprises a synthetic polymer, a naturalpolymer, an injectable gel, a ceramic material, autogeneic tissue,allogeneic tissue, xenogeneic tissue and combinations thereof.
 3. Theimplant of claim 1, wherein the at least one tissue fragment includes aplurality of cells and, upon implantation at a surgical site, at least aportion of the plurality of cells is able to migrate out of the tissuefragment associated with the scaffold to proliferate and integrate withsurrounding tissue at a site of implantation.
 4. The implant of claim 1,wherein the at least one tissue fragment includes a plurality of cellsand, prior to implantation at a surgical site, at least a portion of theplurality of cells is able to migrate out of the tissue fragmentassociated with the scaffold to proliferate and populate the scaffold.5. The implant of claim 1, wherein the biocompatible scaffold furthercomprises an adhesion agent for anchoring the suspension of tissuefragment to the biocompatible scaffold.
 6. The implant of claim 5,wherein the adhesion agent comprises an anchoring agent selected fromthe group consisting of hyaluronic acid, fibrin glue, fibrin clot,collagen gel, gelatin-resorcin-formalin adhesive, mussel-based adhesive,dihydroxyphenylalanine (DOPA) based adhesive, chitosan,transglutaminase, poly(amino acid)-based adhesive, cellulose-basedadhesive, synthetic acrylate-based adhesives, platelet rich plasma(PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA), MonostearoylGlycerol co-Succinate/polyethylene glycol (MGSA/PEG) copolymers,laminin, elastin, proteoglycans and combinations thereof.
 7. The implantof claim 5, wherein the adhesion agent comprises a chemicalcross-linking agent selected from the group consisting of divinylsulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA), formaldehyde,glutaraldehyde, aldehydes, isocyanates, alkyl and aryl halides,imidoesters, N-substituted maleimides, acylating compounds,carbodiimide, hydroxychloride, N-hydroxysuccinimide, light, pH,temperature, and combinations thereof.
 8. The implant of claim 1,wherein the at least one tissue fragment comprises tissue selected fromthe group consisting of cartilage tissue, meniscal tissue, ligamenttissue, tendon tissue, skin tissue, muscle tissue, periosteal tissue,pericardial tissue, synovial tissue, nerve tissue, kidney tissue, bonemarrow, liver tissue, bladder tissue, pancreas tissue, spleen tissue,intervertebral disc tissue, embryonic tissue, periodontal tissue,vascular tissue and combinations thereof.
 9. The implant of claim 8,wherein the at least one tissue fragment comprises autogeneic tissue,allogeneic tissue, xenogeneic tissue, and combinations thereof.
 10. Theimplant of claim 1, where in the at least one tissue fragment comprisesa bone-free tissue type selected from the group consisting of cartilage,meniscus, tendon, ligament and combinations thereof.
 11. The implant ofclaim 1, wherein the biocompatible scaffold comprises a bioabsorbablematerial.
 12. The implant of claim 2, wherein the biocompatible scaffoldcomprises a synthetic polymer selected from the group consisting ofaliphatic polyesters, poly(amino acids), poly(propylene fumarate),copoly(ether-esters), polyalkylene oxalates, polyamides,tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, polyurethanes, biosynthetic polymersand combinations thereof.
 13. The implant of claim 12, wherein thebiocompatible scaffold comprises an aliphatic polyester selected fromthe group consisting of homopolymers or copolymers of lactides;glycolides; ε-caprolactone; hydroxybuterate; hydroxyvalerate;1,4-dioxepan-2-one; 1,5,8,12-tetraoxyacyclotetradecane-7,14-dione;1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine;p-dioxanone (1,4-dioxan-2-one); trimethylene carbonate(1,3-dioxan-2-one); alkyl derivatives of trimethylene carbonate;δ-valerolactone; β-butyrolactone; γ-butyrolactone, ε-decalactone,pivalolactone, α,α-diethylpropiolactone, ethylene carbonate, ethyleneoxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,8-dioxabicycloctane-7-one; andcombinations thereof.
 14. The implant of claim 2, wherein thebiocompatible scaffold comprises a natural polymer selected from thegroup consisting of a fibrin-based material, a collagen-based material,a hyaluronic acid-based material, a cellulose-based material, silk andcombinations thereof.
 15. The implant of claim 2, wherein thebiocompatible scaffold comprises a ceramic material selected from thegroup consisting of hydroxyapatite, α-tricalcium phosphate, β-tricalciumphosphate, bioglass, calcium phospate, calcium carbonate, calciumsulfate, allograft bone graft material, xenograft bone graft materialand combinations thereof.
 16. The implant of claim 1, wherein thebiocompatible scaffold comprises a polymeric foam component having poreswith an open cell pore structure.
 17. The implant of claim 16, whereinthe biocompatible scaffold further comprises a reinforcing componentformed of a biocompatible mesh-containing material.
 18. The implant ofclaim 17, wherein the foam component is integrated with the reinforcingcomponent such that the pores of the foam component penetrate the meshof the reinforcing component and interlock with the reinforcingcomponent.
 19. The implant of claim 1, wherein the biocompatiblescaffold further comprises at least one additional biological componentapplied thereto.
 20. The implant of claim 19, wherein the at least oneadditional biological component comprises growth factors, matrixproteins, peptides, antibodies, enzymes, cytokines, viruses, nucleicacids, peptides, isolated cells, platelets or combinations thereof. 21.The implant of claim 1, wherein the at least one tissue fragment has aparticle size in the range of about 0.1 to 2 mm³.
 22. The implant ofclaim 1, wherein the at least one tissue fragment is added to aphysiological buffering solution to form a suspension having aconcentration of tissue fragments in the range of about 1 to 100 mg/cm².23. The implant of claim 1, wherein the biocompatible implant furthercomprises at least one additional biocompatible scaffold selected fromthe group consisting of a synthetic polymer, a natural polymer, aceramic material, autogeneic tissue, allogeneic tissue, xenogeneictissue and combinations thereof, the at least one additionalbiocompatible scaffold being placed in contact with the at least onetissue fragment, such that at least a portion of the at least one tissuefragment is disposed between at least two biocompatible scaffolds.
 24. Abiocompatible implant, comprising: a biocompatible scaffold; asuspension having at least one cartilage tissue fragment that isassociated with at least a portion of the scaffold, wherein the at leastone tissue fragment in the suspension includes an effective amount ofviable cells that can migrate out of the tissue fragment and populatethe scaffold; and a retaining element, wherein at least a portion of theat least one tissue fragment is disposed between the biocompatiblescaffold and the retaining element.
 25. The implant of claim 24, whereinthe scaffold comprises a synthetic polymer, a natural polymer, aninjectable gel, a ceramic material, autogeneic tissue, allogeneictissue, xenogeneic tissue, and combinations thereof.
 26. The implant ofclaim 24, wherein the retaining element comprises allograft tissueselected from the group consisting of periosteum, perichondrium, fascialata, semitendinosis tendon, gracilis tendon, dura, mesenthera, smallintestine submucosa, skin dermis and combinations thereof.
 27. Theimplant of claim 24, wherein the retaining element is selected from thegroup consisting of autogeneic tissue, allogeneic tissue, xenogeneictissue, a hemostatic material, at least one additional biocompatiblescaffold and combinations thereof
 28. A kit for repairing a tissueinjury, comprising: a sterile container having one or more biocompatiblescaffolds; and a harvesting tool for collecting at least one viabletissue sample from a subject.
 29. The kit of claim 28, furthercomprising at least one reagent for sustaining the viability of the atleast one tissue sample.
 30. The kit of claim 28, wherein the scaffoldis selected from the group consisting of a synthetic polymer, a naturalpolymer, an injectable gel, a ceramic material, autogeneic tissue,allogeneic tissue, xenogeneic tissue, and combinations thereof.
 31. Thekit of claim 28, wherein the harvesting tool further comprises aprocessing tool for dividing the tissue sample, under sterileconditions, into at least one tissue fragment.
 32. The kit of claim 28,wherein the biocompatible scaffold comprises an adhesion agent foranchoring the tissue sample to the biocompatible scaffold.
 33. The kitof claim 32, wherein the adhesion agent comprises an anchoring agentselected from the group consisting of hyaluronic acid, fibrin glue,fibrin clot, collagen gel, alginate gel, gelatin-resorcin-formalinadhesive, mussel-based adhesive, dihydroxyphenylalanine (DOPA) basedadhesive, chitosan, transglutaminase, poly(amino acid)-based adhesive,cellulose-based adhesive, synthetic acrylate-based adhesives, plateletrich plasma (PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG),copolymers, laminin, elastin, proteoglycans and combinations thereof.34. The kit of claim 32, wherein the adhesion agent comprises across-linking agent selected from the group consisting of divinylsulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA), formaldehyde,glutaraldehyde, aldehydes, isocyanates, alkyl and aryl halides,imidoesters, N-substituted maleimides, acylating compounds,carbodiimide, hydroxychloride, N-hydroxysuccinimide, light, pH,temperature, and combinations thereof.
 35. The kit of claim 28, whereinthe at least one reagent comprises a physiological solution selectedfrom the group consisting of saline, phosphate buffer solution, Hank'sbalanced salts, tissue culture medium, tissue culture medium includingserum and combinations thereof.
 36. A method of treating living tissue,comprising: providing a biocompatible scaffold; providing a sample oftissue in the form of finely divided tissue fragments; depositing thesample of tissue upon the biocompatible scaffold to form a tissueimplant; and implanting the tissue implant in a desired positionrelative to the tissue to be treated.
 37. The method of claim 36,wherein the biocompatible scaffold comprises a synthetic polymer, anatural polymer, an injectable gel, a ceramic material, autogeneictissue, allogeneic tissue, xenogeneic tissue, and combinations thereof.38. The method of claim 36, further comprising the step of affixing thetissue implant in the desired position relative to the tissue to betreated.
 39. The method of claim 38, wherein the tissue implant isaffixed in the desired position by applying a fastener to the tissueimplant.
 40. The method of claim 39, wherein the fastener comprises oneor more sutures, one or more staples, one or more suture anchors, one ormore tissue tacks, one or more darts, one or more screws, one or morepins, one or more arrows, fibrin glue, one or more fibrin clots, one ormore biocompatible adhesives or combinations thereof.
 41. The method ofclaim 36, wherein prior to the step of placing the tissue implant in thedesired position relative to the tissue to be treated, the methodfurther includes the step of incubating the tissue implant for aduration and under conditions effective to allow cells within the sampleof tissue to populate the scaffold prior to reimplantation.
 42. Themethod of claim 41, wherein the scaffold and associated finely dividedtissue fragments are incubated for a duration in the range of about 7days to 6 weeks.
 43. The method of claim 41, wherein the scaffold andassociated finely divided tissue fragments are incubated at atemperature in the range of about 20 to 40° C. and in an atmospherehaving a high humidity.
 44. The method of claim 36, wherein the finelydivided tissue fragments comprise tissue selected from the groupconsisting of cartilage tissue, meniscal tissue, ligament tissue, tendontissue, skin tissue, muscle tissue, periosteal tissue, pericardialtissue, synovial tissue, nerve tissue, kidney tissue, bone marrow, livertissue, bladder tissue, pancreas tissue, spleen tissue, and combinationsthereof.
 45. The method of claim 44, wherein the finely divided tissuefragments comprise autogeneic tissue, allogeneic tissue, xenogeneictissue, and combinations thereof.
 46. The implant of claim 36, where inthe finely divided tissue fragments comprise a bone-free tissue typeselected from the group consisting of cartilage, meniscus, tendon,ligament and combinations thereof.
 47. The method of claim 36, whereinthe finely divided tissue fragments associated with the biocompatiblescaffold comprise a type that is the same as the tissue to be treated.48. The method of claim 36, wherein the finely divided tissue fragmentsassociated with the biocompatible scaffold comprise a type that isdifferent from the tissue to be treated.
 49. The method of claim 36,wherein the finely divided tissue fragments include an effective amountof viable cells that can migrate out of the tissue particles.
 50. Themethod of claim 49, wherein the effective amount of cells migrate out ofthe tissue particles and populate an outer surface of the biocompatiblescaffold.
 51. The method of claim 49, wherein the effective amount ofcells migrate out of the tissue particles and populate at least aportion of an interior region of the scaffold, such that the cells areembedded within the scaffold.
 52. The method of claim 36, wherein themethod further comprises, prior to placing the tissue implant in thedesired position relative to the tissue to be treated, the additionalstep of providing at least one additional biocompatible scaffold andplacing the at least one additional biocompatible scaffold over thedeposited finely divided tissue fragments, such that at least a portionof the finely divided tissue fragments is disposed between at least twobiocompatible scaffolds.
 53. The method of claim 36, wherein thebiocompatible scaffold further comprises an adhesion agent for anchoringthe sample of living tissue to the biocompatible scaffold.
 54. Themethod of claim 53, wherein the adhesion agent comprises an anchoringagent selected from the group consisting of hyaluronic acid, fibringlue, fibrin clot, collagen gel, alginate gel, gelatin-resorcin-formalinadhesive, mussel-based adhesive, dihydroxyphenylalanine (DOPA) basedadhesive, chitosan, transglutaminase, poly(amino acid)-based adhesive,cellulose-based adhesive, synthetic acrylate-based adhesives, plateletrich plasma (PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG)copolymers, laminin, elastin, proteoglycans and combinations thereof.55. The method of claim 53, wherein the adhesion agent comprises across-linking agent selected from the group consisting of divinylsulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA), formaldehyde,glutaraldehyde, aldehydes, isocyanates, alkyl and aryl halides,imidoesters, N-substituted maleimides, acylating compounds,carbodiimide, hydroxychloride, N-hydroxysuccinimide, light, pH,temperature, and combinations thereof.
 56. The method of claim 37,wherein the biocompatible scaffold comprises a bioabsorbable material.57. The method of claim 37, wherein the biocompatible scaffold comprisesa synthetic polymer selected from the group consisting of aliphaticpolyesters, poly(amino acids), copoly(ether-esters), polyalkyleneoxalates, polyamides, tyrosine-derived polycarbonates,poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups, poly(anhydrides),polyphosphazenes, polyurethanes, biosynthetic polymers and combinationsthereof.
 58. The method of claim 57, wherein the biocompatible scaffoldcomprises an aliphatic polyester selected from the group consisting ofhomopolymers or copolymers of lactides; glycolides; ε-caprolactone;hydroxybuterate; hydroxyvalerate; 1,4-dioxepan-2-one;1,5,8,12-tetraoxyacyclotetradecane-7,14-dione; 1,5-dioxepan-2-one;6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine; p-dioxanone(1,4-dioxan-2-one); trimethylene carbonate (1,3-dioxan-2-one); alkylderivatives of trimethylene carbonate; δ-valerolactone; β-butyrolactone;γ-butyrolactone; ε-decalactone; pivalolactone; α,α-diethylpropiolactone;ethylene carbonate; ethylene oxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,8-dioxabicycloctane-7-one; andcombinations thereof.
 59. The method of claim 37, wherein thebiocompatible scaffold comprises a natural polymer selected from thegroup consisting of a fibrin-based material, a collagen-based material,a hyaluronic acid-based material, a cellulose-based material, silk andcombinations thereof.
 60. The method of claim 37, wherein thebiocompatible scaffold comprises a ceramic selected from the groupconsisting of hydroxyapatite, α-tricalcium phosphate, β-tricalciumphosphate, calcium phosphate, calcium carbonate, calcium sulfate,bioglass, allogeneic bone graft material, xenogeneic bone graft materialand combinations thereof.
 61. The method of claim 36, wherein thebiocompatible scaffold comprises a polymeric foam component having poreswith an open cell pore structure.
 62. The method of claim 61, whereinthe biocompatible scaffold further comprises a reinforcing componentformed of a biocompatible mesh-containing material.
 63. The method ofclaim 62, wherein the foam component is integrated with the reinforcingcomponent such that the pores of the foam component penetrate the meshof the reinforcing component and interlock with the reinforcingcomponent.
 64. The method of claim 36, wherein the biocompatiblescaffold further comprises at least one additional biological componentapplied thereto.
 65. The method of claim 64, wherein the at least oneadditional biological component comprises growth factors, matrixproteins, enzymes, cytokines, viruses, nucleic acids, peptides, isolatedcells, platelets or combinations thereof.
 66. The method of claim 36,wherein the finely divided, minced tissue particles further comprise aplurality of cells, and at least a portion of the plurality of cells aretransfected or transduced using a vector including at least one gene.67. The method of claim 65, wherein the vector comprises a viral vectoror a non-viral vector.
 68. The method of claim 66, wherein the at leastone gene encodes a gene product of interest.
 69. The method of claim 68,wherein the gene product of interest comprises proteins, polypeptides,interference ribonucleic acid (iRNA) or combinations thereof.
 70. Themethod of claim 36, wherein the method of treating tissue is a tissuetreatment technique selected from the group consisting of tissue repair,tissue bulking, cosmetic treatment, therapeutic treatment, tissueaugmentation, and tissue sealing.
 71. A method of preparing a tissueimplant, comprising: providing a bioimplantable scaffold; obtaining asample of tissue; processing the sample of tissue under asepticconditions to form at least one tissue fragment and a physiologicalbuffering solution; and depositing the tissue fragment on thebioimplantable scaffold to yield a tissue implant.
 72. The method ofclaim 71, wherein the bioimplantable scaffold comprises a syntheticpolymer, a natural polymer, an injectable gel, a ceramic material,autogeneic tissue, allogeneic tissue, xenogeneic tissue, andcombinations thereof.
 73. The method of claim 71, wherein the methodfurther comprises the step of incubating the tissue implant for aduration and under conditions effective to allow cells within the atleast one tissue fragment to populate the scaffold.
 74. The method ofclaim 71, wherein the tissue implant is incubated for a duration in therange of about 7 days to 6 weeks.
 75. The method of claim 74, whereinthe tissue implant is incubated at a temperature in the range of about20 to 40° C. and in an atmosphere having high humidity.
 76. The methodof claim 71, wherein the at least one tissue fragment comprises tissueselected from the group consisting of cartilage tissue, meniscal tissue,ligament tissue, tendon tissue, skin tissue, muscle tissue, periostealtissue, pericardial tissue, synovial tissue, nerve tissue, kidneytissue, bone marrow, liver tissue, bladder tissue, pancreas tissue,spleen tissue, and combinations thereof.
 77. The method of claim 76,wherein the at least one tissue fragment comprises autologous tissue.78. The implant of claim 71, where in the at least one tissue fragmentcomprises a bone-free tissue type selected from the group consisting ofcartilage, meniscus, tendon, ligament and combinations thereof.
 79. Themethod of claim 71, wherein the at least one tissue fragment comprisesan effective amount of viable cells that can migrate out of the tissuefragment.
 80. The method of claim 79, wherein the effective amount ofcells migrate out of the tissue fragment and populate an outer surfaceof the bioimplantable scaffold.
 81. The method of claim 79, wherein theeffective amount of cells migrate out of the tissue fragment andpopulate at least a portion of an interior region of the scaffold, suchthat the cells are embedded within the scaffold.
 82. The method of claim71, further comprising the additional step of providing at least oneadditional bioimplantable scaffold and placing the at least oneadditional bioimplantable scaffold over the deposited at least onetissue fragment, such that at least a portion of the at least one tissuefragment is disposed between at least two bioimplantable scaffolds. 83.The method of claim 71, wherein the bioimplantable scaffold furthercomprises an adhesion agent for anchoring the at least one minced tissuefragment to the bioimplantable scaffold.
 84. The method of claim 83,wherein the adhesion agent comprises an anchoring agent selected fromthe group consisting of hyaluronic acid, fibrin glue, fibrin clot,collagen gel, alginate gel, gelatin-resorcin-formalin adhesive,mussel-based adhesive, dihydroxyphenylalanine (DOPA) based adhesive,chitosan, transglutaminase, poly(amino acid)-based adhesive,cellulose-based adhesive, synthetic acrylate-based adhesives, plateletrich plasma (PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG)copolymers, laminin, elastin, proteoglycans and combinations thereof.85. The method of claim 83, wherein the adhesion agent comprises across-linking agent selected from the group consisting of divinylsulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA), formaldehyde,glutaraldehyde, aldehydes, isocyanates, alkyl and aryl halides,imidoesters, N-substituted maleimides, acylating compounds,carbodiimide, hydroxychloride, N-hydroxysuccinimide, light, pH,temperature, and combinations thereof.
 86. The method of claim 71,wherein the bioimplantable scaffold comprises a bioabsorbable material.87. The method of claim 72, wherein the bioimplantable scaffoldcomprises a synthetic polymer selected from the group consisting ofaliphatic polyesters, poly(amino acids), poly(propylene fumarate),copoly(ether-esters), polyalkylene oxalates, polyamides,tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, polyurethanes, biosynthetic polymersand combinations thereof.
 88. The method of claim 87, wherein thebiocompatible scaffold comprises an aliphatic polyester selected fromthe group consisting of homopolymers or copolymers of lactides;glycolides; ε-caprolactone; hydroxybuterate; hydroxyvalerate;1,4-dioxepan-2-one; 1,5,8,12-tetraoxyacyclotetradecane-7,14-dione;1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine;p-dioxanone (1,4-dioxan-2-one); trimethylene carbonate(1,3-dioxan-2-one); alkyl derivatives of trimethylene carbonate;δ-valerolactone; β-butyrolactone; γ-butyrolactone; ε-decalactone;pivalolactone; α,α-diethylpropiolactone; ethylene carbonate; ethyleneoxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,8-dioxabicycloctane-7-one; andcombinations thereof.
 89. The method of claim 72, wherein thebioimplantable scaffold comprises a natural polymer selected from thegroup consisting of a fibrin-based material, a collagen-based material,a hyaluronic acid-based material, a cellulose-based material, silk andcombinations thereof.
 90. The method of claim 72, wherein thebioimplantable scaffold comprises a ceramic selected from the groupconsisting of hydroxyapatite, α-tricalcium phosphate, β-tricalciumphosphate, bioglass, allogeneic bone graft material, xenogeneic bonegraft material and combinations thereof.
 91. The method of claim 71,wherein the bioimplantable scaffold comprises a polymeric foam componenthaving pores with an open cell pore structure.
 92. The method of claim91, wherein the bioimplantable scaffold further comprises a reinforcingcomponent formed of a biocompatible mesh-containing material.
 93. Themethod of claim 92, wherein the foam component is integrated with thereinforcing component such that the pores of the foam componentpenetrate the mesh of the reinforcing component and interlock with thereinforcing component.
 94. The method of claim 71, wherein thebioimplantable scaffold further comprises at least one additionalbiological component applied thereto.
 95. The method of claim 94,wherein the at least one additional biological component comprisesgrowth factors, matrix proteins, enzymes, cytokines, viruses, nucleicacids, peptides, isolated cells, platelets or combinations thereof. 96.A method for measuring the effect of a substance on living tissue,comprising the steps of: (a) creating a tissue construct by providing abiocompatible scaffold, obtaining a sample of tissue, processing thesample of tissue to form at least one tissue fragment, depositing the atleast one tissue fragment on the biocompatible scaffold such that the atleast one tissue fragment is associated with the biocompatible scaffold,thereby forming a tissue construct, and incubating the tissue constructfor a duration and under conditions that are effective to allow cellswithin the tissue fragment to populate the scaffold; (b) contacting thetissue construct with a substance; and (c) determining the effects ofthe substance on the tissue construct.
 97. The method of claim 96,wherein the substance comprises a drug, a pharmaceutical composition, achemical, a microbe, an element, a cytokine, a growth factor, a hormone,an antibody, a peptide, a ligand, an antagonist of membrane-boundreceptors, or combinations thereof.